Anti-ptk7 antibody-drug conjugates

ABSTRACT

The present invention provides anti-PTK7 antibody-drug conjugates and methods for preparing and using the same.

RELATED APPLICATION

This is a continuation of U.S. patent application Ser. No. 14/696,663filed Apr. 27, 2015, which claims priority to U.S. Provisional PatentApplication No. 61/986,520, filed Apr. 30, 2014, each of which isincorporated by reference herein in its entirety.

SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“PC072045_Sequence_Listing.txt” created on Apr. 30, 2014, and having asize of 57.7 KB. The sequence listing contained in this .txt file ispart of the specification and which is incorporated herein by referencein its entirety.

FIELD OF THE INVENTION

The present invention relates to protein tyrosine kinase 7 (PTK7)antibodies and antibody-drug conjugates. The present invention furtherrelates to the methods of using such antibodies and antibody-drugconjugates for the treatment of cancer.

BACKGROUND OF THE INVENTION

Protein tyrosine kinase 7(PTK7), also known as colon carcinoma kinase 4(CCK4), is a receptor tyrosine kinase originally cloned from normalhuman melanocytes and separately from colon carcinoma tissue. Highlevels of PTK7 have been identified in a number of tumor cells,including bladder, breast, colorectal, kidney, and lung cancers andmelanoma. PTK7 expression has also been observed on adult myeloidleukemia (AML) and acute lymphoblastic leukemia (ALL) cells.

The treatment of cancer has improved over the past decade with surgery,radiation therapy, and chemotherapy as the primary treatment options.Such treatments can extend survival and/or relieve symptoms in manypatients but are not likely to produce a cure for many patients.Consequently, there remains a significant need for additionaltherapeutic options for cancers.

To this end, the present invention provides novel antibody-drugconjugates that target PTK7-positive cancers. The disclosed anti-PTK7antibody-drug conjugates can exert a clinically useful cytotoxic effecton PTK7 expressing tumor cells without exerting undesirable effects onnon-PTK7 expressing cells.

SUMMARY OF THE INVENTION

The present invention provides PTK7 antibody-drug conjugates and theiruse in detection, prophylaxis, and therapy of PTK7 associated disorders.A PTK7 antibody-drug conjugate of the invention is generally of theformula: Ab-(L-D), wherein Ab is an antibody, or antigen-bindingfragment thereof, that binds to PTK7, or a PTK7-binding fragmentthereof; and L-D is a linker-drug moiety, wherein L is a linker, and Dis a drug.

The Ab of the disclosed antibody-drug conjugate can be any PTK7-bindingantibody. In some aspects of the invention, the Ab is a chimeric,CDR-grafted, humanized, or a recombinant human antibody, or PTK7-bindingfragment thereof. In some aspects of the invention, the Ab is aninternalizing antibody and/or a neutralizing antibody.

The present invention also provides PTK7 antibody-drug conjugates andtheir use in detection, prophylaxis and therapy of PTK7 associateddisorders. A PTK7 antibody-drug conjugate of the invention is generallyof the formula: Ab-(L-D), wherein Ab is an antibody, or antigen-bindingfragment thereof, that binds to PTK7, or a PTK7-binding fragmentthereof; and L-D is a linker-drug moiety, wherein L is a linker, and Dis an auristatin.

In particular aspects of the invention, the Ab is a hu23, hu24, or hu58antibody, or an antibody that competes for binding to human PTK7 withhu23, hu24, or hu58, and/or an antibody that binds to the same epitopeas a hu23, hu24, or hu58 antibody. For example, the Ab may compete forbinding to human PTK7 with, and/or bind the same epitope as, an antibodycomprising (a) a heavy chain variable region set forth as SEQ ID NO: 1and a light chain variable region set forth as SEQ ID NO: 15; (b) aheavy chain variable region set forth as SEQ ID NO: 25 and a light chainvariable region set forth as SEQ ID NO: 39; or (c) a heavy chainvariable region set forth as SEQ ID NO: 49 and a light chain variableregion set forth as SEQ ID NO: 63.

Among Abs that compete for binding to human PTK7 with hu23, and/or bindto the same epitope as hu23, representative Abs useful for preparingPTK7 antibody-drug conjugates of the invention include antibodiescomprising at least one heavy chain variable region and at least onelight chain variable region, wherein the at least one heavy chainvariable region comprises three CDRs defined by SEQ ID NOs: 3, 7, and11. Additional Abs include antibodies comprising at least one heavychain variable region and at least one light chain variable region,wherein the at least one light chain variable region comprises threeCDRs defined as SEQ ID NOs: 17, 19, and 21. Additional Abs includeantibodies comprising (a) a heavy chain variable region comprising threeCDRs set forth as SEQ ID NOs: 3, 7, and 11; and (b) a light chainvariable region comprising three CDRs set forth as SEQ ID NOs: 17, 19,and 21.

In other PTK7 antibody-drug conjugates of the invention, the Abcomprises a heavy chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 1 and a light chainvariable having an amino acid sequence that is at least 90% identical toSEQ ID NO: 15, for example, a heavy chain variable region set forth asSEQ ID NO: 1 and a light chain variable region set forth as SEQ ID NO:15.

Among Abs that compete for binding to human PTK7 with hu24, and/or bindto the same epitope as hu24, representative Abs useful for preparingPTK7 antibody-drug conjugates of the invention include antibodiescomprising at least one heavy chain variable region and at least onelight chain variable region, wherein the at least one heavy chainvariable region comprises three CDRs defined by SEQ ID NOs: 27, 31, and35. Additional Abs include antibodies comprising at least one heavychain variable region and at least one light chain variable region,wherein the at least one light chain variable region comprises threeCDRs defined as SEQ ID NOs: 41, 43, and 45. Additional Abs includeantibodies comprising (a) a heavy chain variable region comprising threeCDRs set forth as SEQ ID NOs: 27, 31, and 35; and (b) a light chainvariable region comprising three CDRs set forth as SEQ ID NOs: 41, 43,and 45.

In other PTK7 antibody-drug conjugates of the invention, the Abcomprises a heavy chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 25 and a light chainvariable having an amino acid sequence that is at least 90% identical toSEQ ID NO: 39, for example, a heavy chain variable region set forth asSEQ ID NO: 25 and a light chain variable region set forth as SEQ ID NO:39.

Among Abs that compete for binding to human PTK7 with hu58, and/or bindto the same epitope as hu58, representative Abs useful for preparingPTK7 antibody-drug conjugates of the invention include antibodiescomprising at least one heavy chain variable region and at least onelight chain variable region, wherein the at least one heavy chainvariable region comprises three CDRs defined by SEQ ID NOs: 51, 55, and59. Additional Abs include antibodies comprising at least one heavychain variable region and at least one light chain variable region,wherein the at least one light chain variable region comprises threeCDRs defined as SEQ ID NOs: 65, 67, and 69. Additional Abs includeantibodies comprising (a) a heavy chain variable region comprising threeCDRs set forth as SEQ ID NOs: 51, 55, and 59; and (b) a light chainvariable region comprising three CDRs set forth as SEQ ID NOs65, 67, and69.

In other PTK7 antibody-drug conjugates of the invention, the Abcomprises a heavy chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 49 and a light chainvariable having an amino acid sequence that is at least 90% identical toSEQ ID NO: 63, for example, a heavy chain variable region set forth asSEQ ID NO: 49 and a light chain variable region set forth as SEQ ID NO:63.

In some aspects of the invention, PTK7 antibody-drug conjugates comprisean Ab comprising an IgG1 heavy chain constant region, a kappa lightchain constant region, or an IgG1 heavy chain constant region and akappa light chain constant region. For example, Abs useful for preparingPTK7 antibody-drug conjugates of the invention include antibodiescomprising a heavy chain set forth as SEQ ID NO: 13, a light chain setforth as SEQ ID NO: 23, or a heavy chain set forth as SEQ ID NO: 13 anda light chain set forth as SEQ ID NO: 23. Additional examples includeantibodies comprising a heavy chain set forth as SEQ ID NO: 37, a lightchain set forth as SEQ ID NO: 47, or a heavy chain set forth as SEQ IDNO: 37 and a light chain set forth as SEQ ID NO: 47. Still furtherexamples include antibodies comprising a heavy chain set forth as SEQ IDNO: 61, a light chain set forth as SEQ ID NO: 71, or a heavy chain setforth as SEQ ID NO: 61 and a light chain set forth as SEQ ID NO: 71.

In other aspects of the invention, a PTK7 antibody-drug conjugate of theinvention comprises an antibody having a heavy chain variable region setforth as SEQ ID NO: 1, 25, or 49. In other aspects of the invention, aPTK7 antibody-drug conjugate of the invention comprises an antibodyhaving light chain variable region set forth as SEQ ID NO: 15, 39, or63.

In particular aspects of the invention, the Ab is a hu23, hu24 or hu58antibody, or an antibody that competes for binding to human PTK7 withhu23, hu24 or hu58 antibody and/or an antibody that binds to the sameepitope as hu23, hu24 or hu58 antibody. For example, the Ab may competefor binding to human PTK7 with and/or bind to the same epitope as anantibody comprising (a) a heavy chain variable region of SEQ ID NO:13and a light chain variable region of SEQ ID NO:23; (b) a heavy chainvariable region of SEQ ID NO:25 and a light chain variable region of SEQID NO:39; or (c) a heavy chain variable region of SEQ ID NO:49 and alight chain variable region of SEQ ID NO:63.

In another aspect of the invention, the Ab is a humanized monoclonalantibody such as hu23, hu24 or hu58 antibody.

Any of the PTK7 antibody-drug conjugates disclosed herein may beprepared with a linker that is cleavable or non-cleavable. In one aspectthe cleavable linker may bemaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc). Inanother aspect the cleavable linker may be 4 (4′acetylphenoxy)butanoicacid (AcBut). In another aspect the non-cleavable linker may bemaleimidocaproyl (mc).

Any of the PTK7 antibody drug conjugates disclosed herein may beprepared with a drug that is auristatin. In one aspect, the auristatinmay be 0101(2-Methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).In another aspect, the auristatin may be 82612-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.

Any of the PTK7 antibody drug conjugates disclosed herein may beprepared with a drug that is calicheamicin, including N acetylderivatives of calicheamicin such as N-acetyl-γ-calicheamicin andN-acetyl-γ-calicheamicin dimethyl hydrazide (CM).

Any of the PTK7 antibodies disclosed herein may be used in anantibody-drug conjugate by conjugation with a linker-drug moiety (L-D).In one aspect, the L-D may be vc0101(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[(21S,24S,25R)-24-[(2S)-butan-2-yl]-25-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-18,18,23-trimethyl-3,16,19,22-tetraoxo-21-(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N˜5˜-carbamoyl-L-ornithinamide).In another aspect, the L-D may be mc8261(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).In yet another aspect, the L-D may be AcButCM (4(4′acetylphenoxy)butanoic acid N-acetyl-γ-calicheamicin dimethylhydrazide). Any of the PTK7 antibody-drug conjugates disclosed hereinmay have a drug-to-antibody ratio (DAR) from 1 to 8.

In a particular aspect of the invention, a PTK7 antibody-drug conjugateof the formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:13 and a light chain set forth as SEQ ID NO: 23; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:37 and a light chain set forth as SEQ ID NO: 47; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:61 and a light chain set forth as SEQ ID NO: 71; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:13 and a light chain set forth as SEQ ID NO: 23; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl (mc), and wherein the drug is 8261.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:37 and a light chain set forth as SEQ ID NO: 47; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl (mc), and wherein the drug is 8261.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:61 and a light chain set forth as SEQ ID NO: 71; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl (mc), and wherein the drug is 8261.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:13 and a light chain set forth as SEQ ID NO: 23; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris AcBut (4 (4′acetylphenoxy)butanoic acid) and wherein the drug is CM(N-acetyl-γ-calicheamicin dimethyl hydrazide).

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:37 and a light chain set forth as SEQ ID NO: 47; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris AcBut (4 (4′acetylphenoxy)butanoic acid) and wherein the drug is CM(N-acetyl-γ-calicheamicin dimethyl hydrazide).

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:61 and a light chain set forth as SEQ ID NO: 71; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris AcBut (4 (4′acetylphenoxy)butanoic acid) and wherein the drug is CM(N-acetyl-γ-calicheamicin dimethyl hydrazide).

The present invention provides for compositions comprising a pluralityof antibody-drug conjugates disclosed herein and optionally apharmaceutical carrier, wherein the composition has an average DARwithin the range of 1 to 8. In a particular aspect of the invention, thecomposition may have an average DAR within the range of 3 to 5. Inanother aspect of the invention, the composition may have an average DARwithin the range of 3 to 4. In another aspect of the invention, thecomposition may have an average DAR of about 4.

The present invention further provides for a composition comprising aplurality of an antibody-drug conjugate disclosed herein and optionallya pharmaceutical carrier, wherein the composition has at least 50%antibody-drug conjugates having a DAR from 3 to 5. In another aspect ofthe invention, the composition has at least 60% antibody-drug conjugateshaving a DAR from 3 to 5.

The present invention further provides for a PTK7 antibody-drugconjugate that is generally of the formula: Ab-(L-D), wherein Ab is anantibody or antigen-binding fragment thereof that binds to PTK7 or aPTK7-binding fragment thereof; and L-D is a linker-drug moiety, whereinL is vc or mc or AcBut, and D is a drug.

The present invention further provides for a PTK7 antibody-drugconjugate that is generally of the formula: Ab-(L-D), wherein Ab is anantibody, or antigen-binding fragment thereof that binds to PTK7, or aPTK7-binding fragment thereof; and L-D is a linker-drug moiety, whereinL is a linker, and D is an auristatin (such as 0101 or 8261) or CM.

The present invention further provides methods for preparing a PTK7antibody-drug conjugate disclosed herein. For example, a process forproducing an antibody-drug conjugate can include the steps of (a)linking the linker to the drug; (b) conjugating the linker-drug moietyto the antibody; and (c) purifying the antibody-drug conjugate.

Another aspect of the invention includes methods of making, methods ofpreparing, methods of synthesis, methods of conjugation and methods ofpurification of the antibody-drug conjugates disclosed herein and theintermediates for the preparation, synthesis and conjugation of theantibody-drug conjugates disclosed herein.

Further provided are pharmaceutical compositions comprising a PTK7antibody-drug conjugate disclosed herein and a pharmaceuticallyacceptable carrier.

In other aspects are provided methods of treating a PTK7 associateddisorder by administering a therapeutically effective amount of acomposition comprising a PTK7 antibody-drug conjugate disclosed herein.Representative PTK7 associated disorders include hyperproliferativedisorders, such as neoplastic disorders, such as solid tumors (e.g.,breast cancer, such as triple-negative breast cancer (TNBC),progesterone-receptor positive breast cancer (PR+), estrogen-receptorpositive breast cancer (ER+) and double positive breast cancer; ovariancancer; colorectal cancer; esophageal cancer; gastric cancer; melanoma;sarcoma; kidney cancer; pancreatic cancer; prostate cancer; livercancer, such as hepatocellular carcinoma (HCC); and lung cancer, such asnon-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC),etc.) and hematologic malignancies (e.g., leukemia, such as adultmyeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), etc.).Also provided are uses of the disclosed PTK7 antibody-drug conjugatesfor the manufacture of a medicament for treating a PTK7 associateddisorder in a subject. Also provided are PTK7 antibody-drug conjugatesfor use in the treatment of a PTK7 associated disorder.

In other aspects, the present invention provides for methods of treatinga PTK7 associated disorder in a subject by administering atherapeutically effective amount of a composition comprising a PTK7antibody-drug conjugate disclosed herein and a chemotherapeutic agent.

Another aspect of the invention includes methods of treating a disordercharacterized by the overexpression of PTK7 in a patient with anantibody-drug conjugate disclosed herein. In other aspects, the presentinvention provides for methods of treating cancer characterized by theoverexpression of PTK7 in a patient with an antibody-drug conjugatedisclosed herein.

In still other aspects, the present invention provides a method ofreducing tumor initiating cells in a tumor cell population. For example,the method can comprise contacting a tumor cell population, wherein thepopulation comprises tumor initiating cells and tumor cells other thantumor initiating cells, with a PTK7 antibody-drug conjugate; whereby thefrequency of tumor initiating cells in the tumor cell population isreduced. The contacting step may be performed in vitro or in vivo.

Another aspect of the invention includes diagnostic and therapeutic usesfor the compounds and compositions disclosed herein.

Other aspects of the invention include articles of manufacture, i.e.kits, comprising an antibody-drug conjugate disclosed herein, acontainer, and a package insert or label indicating a treatment.

These and other aspects of the invention will be appreciated by a reviewof the application as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid sequence of a representative full-lengthPTK7 protein (SEQ ID NO. 73).

FIGS. 2A-B show that hu24 binding correlates with cell expression ofPTK7. (A) Immunoblot of whole cell lysates with anti-PTK7 and anti-GAPDHantibodies. The immunoblot signal had been previously validated bydemonstrating loss of signal when PTK7 gene expression was inhibited bysiRNA. (B) Mean fluorescence intensity values from flow cytometry withanti-PTK7 (hu24) on the same cancer cell lines as in the immunoblot. Thedotted line represents the signal when the primary antibody was anegative control antibody rather than hu24.

FIG. 3 shows PTK7 expression on tissue samples from seven PDX models byimmunohistochemistry. Staining indicates PTK7. Representativemicrographs are shown for each model. Scale bar, 100 μM.

FIGS. 4A-C are graphs showing PTK7 mRNA expression in primary tumors.(A) breast cancers; (B) NSCLC cancers and (C) ovarian cancer are shown.

FIG. 5 is a graph showing a correlation between higher PTK7 mRNAexpression and worse overall survival rates in NSCLC patients.

FIG. 6 is a graph showing PTK7 protein levels in serum from healthyhumans and cancer patients representing 8 different tumor types. Thehorizontal lines indicate the mean value for each group.

FIG. 7 provides the hydrophobic interaction chromatography (HIC)analysis of hu24-vc0101.

FIG. 8 is a graph showing the efficacy of anti-PTK7-vc0101 ADCs in theBreast-13 (BR13) triple-negative breast cancer (TNBC) PDX.

FIG. 9 is a data table showing the efficacy of hu24-vc0101 andanti-PTK7-mc8261 ADCs in the BR13 TNBC PDX.

FIG. 10 is a graph of the data of FIG. 9 showing the efficacy ofhu24-vc0101 and anti-PTK7-mc8261 ADCs in the BR13 TNBC PDX.

FIG. 11 is a graph showing the efficacy of anti-PTK7-vc0101 ADCs in theBreast-22 (BR22) TNBC PDX.

FIG. 12 is a data table showing the efficacy of hu23-vc0101 andanti-PTK7-mc8261 ADCs in the BR22 TNBC PDX.

FIG. 13 is a graph of the data of FIG. 12 showing the efficacy ofhu23-vc0101 and anti-PTK7-mc8261 ADCs in the BR22 TNBC PDX.

FIG. 14 is graph showing the efficacy of anti-PTK7-vc0101 ADCs in theBreast-31 (BR31) TNBC PDX.

FIG. 15 is a graph showing the efficacy of hu24-vc0101 ADC in theBreast-64 (BR64) TNBC PDX.

FIG. 16 is a graph showing the efficacy of hu24-vc0101 ADC in the BR5TNBC PDX.

FIG. 17 is a graph showing the efficacy of hu24-vc0101 ADC in the BR36PR+TNBC PDX.

FIGS. 18A-B is a graph showing the efficacy of hu24-vc0101 andhu23-AcBut CM in two different SCLC PDX models (A) H1048 PDX model and(B) SCLC 95 PDX model.

FIGS. 19A-B is a graph showing the efficacy of hu24-AcButCM in twodifferent SCLC PDX models (A) a SCLC 117 PDX model and (B) a SCLC 102PDX model.

FIG. 20 is a graph showing the efficacy of hu24-vc0101 ADC in theLung-135 (LU135) non-small cell lung cancer (NSCLC) PDX.

FIG. 21 is a graph showing the efficacy of hu24-vc0101 ADC in theLung-176 (LU176) non-small cell lung cancer (NSCLC) PDX.

FIG. 22 shows micrographs of microtubule structure after treatment with4 g/mL hu24-vc0101 ADC, 4 g/mL unconjugated hu24 mAb, 4 g/mL negativecontrol ADC, or 0.1 nM free 0101 auristatin. H661 cells were treated for48 hours and then stained for anti-tubulin and DAPI to visualize theDNA.

FIG. 23 shows the effect of hu24-vc0101 ADC on endothelial cells invitro.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides antibody-drug conjugates that bind toPTK7, processes for preparing the conjugates using PTK7 antibodies,linkers, and drugs. The antibody-drug conjugates of the invention areuseful for the preparation and manufacture of compositions, such asmedicaments that may be used in the diagnosis, prophylaxis, and/ortreatment of hyperproliferative disorders characterized by PTK7expression. In some aspects of the invention, the disclosedantibody-drug conjugates may reduce the frequency of tumor initiatingcells (TIC), which encompass both tumor perpetuating cells (TPC) andhighly proliferative tumor progenitor cells (TProg).

I. PTK7 Physioloqy

Protein tyrosine kinase (PTK7), also known as colon carcinoma kinase 4(CCK4), is a receptor tyrosine kinase originally cloned from normalhuman melanocytes (Lee et al., Oncogene 8(12):3403-3410, 1993) andseparately from colon carcinoma tissue (Mossie et al., Oncogene11(10):2179-2184, 1995). The PTK7 gene is located at 6p21.1-p12.2. Fivesplice isoforms of human PTK7 have been cloned from testis cDNA (Jung,et al., Biochim Biophys Acta 1579, 2002). The relative abundance of theisoforms with respect to one another differs between testis and hepatomaor colon carcinoma lines, but the functional significance of theseisoforms, if any, is unknown. Bioinformatics analyses have suggestedthat the mouse may express a soluble PTK7 isoform from alternativelyspliced mRNAs (Forrest, Taylor et al., Genome Biol 7, 2006).

Full length PTK7 protein is a type I transmembrane protein, with a 674amino acid extracellular domain (ECD), followed by a short TM spanningportion and a 345 amino acid cytoplasmic domain. A representative afull-length amino acid sequence of PTK7 is shown in FIG. 1 (SEQ ID NO.73). The amino acid sequences of representative PTK7 isoforms are foundin GenBank Accession Nos. EAX04154.1 (isoform a), EAX04155.1 (isoformb), EAX04156.1 (isoform c), EAX04157.1 (isoform d), EAX04158.1 (isoforme), EAX04159.1 (isoform f), and EAX04160.1 (isoform g). All isoformsencode the same intracellular domain. A complete nucleic acid sequenceof a representative isoform of human PTK7 (i.e. transcript variantPTK7-1), has Genbank Accession No. NM_002821.

The mature full length PTK7 ECD comprises seven immunoglobulin-likedomains while the various splice variants encode PTK7 isoforms thatdiffer in their ECD structure. All isoforms contain a cytoplasmic domainwith substantial homology to that found in the general class of tyrosinekinases. However, PTK7 lacks detectable tyrosine kinase activity and, assuch, belongs to a subfamily of pseudokinases in which several aminoacid changes in various conserved kinase subdomains lead to impairedbinding of ATP (Boudeau, et al., Trends Cell Biol. 16, 2006).Specifically, key residues in subdomains I and VII are altered in PTK7such that direct interactions with the non-transferable phosphates ofATP, as well as, chelation of the Mg²⁺ cofactor bridging thesephosphates, would be impaired.

The biological importance of PTK7 function can be inferred from thepresence of conserved orthologs from Hydra through Drosophila to chickenand human, each of which by sequence analysis is predicted to lackkinase activity. Based upon the high conservation of a specific TMdomain motif associated with a propensity for helix-helix association,it has been suggested that the TM domain may mediate PTK7 dimerization.The PTK7 pseudokinase domain itself is not expected to directly transmitthe signal, but it may interact as a scaffold for other molecules in thesignaling pathway, or may recruit other tyrosine kinase(s). It has beenshown that PTK7 may function in cell adhesion, cell migration, cellpolarity, proliferation, actin cytoskeleton reorganization, andapoptosis to regulate embryogenesis, epithelial tissue organization,neuronal tube closure, neuronal crest formation, and axon guidance(Peradziryi, H. et al. Arch Biochem Biophys. 524, 2012).

Normal tissues and cells reported to express PTK7 include lung, thyroid,ovary, CD4+ recent thymic emigrant T-cells, and normal myeloidprogenitors and CD34+CD38− bone marrow cells. With respect to canceroustissues, PTK7 expression has also been found in colon carcinoma cells,adult myeloid leukemia (AML) samples, CD34− pre-TALL cells, and gastriccarcinoma. PTK7 may be lost in certain breast cancers containingdeletions of chromosome 6p21, although expression is variable in breastcancer cell lines. PTK7 is also expressed in lung adenocarcinoma. Finemapping of the amplifications of 6pl 2-p21 region in osteosarcomas hasshown that increases in gene copy number do not necessarily result inoverexpression of PTK7, as determined by qRT-PCR.

The ligand or ligands for PTK7 are not known, although PTK7 has beenlinked to a variety of biological signaling pathways and developmentalprocesses. For example, PTK7 acts as a co-receptor in both thenon-canonical (also known as the Wnt/planar cell polarity signaling) andthe canonical Wnt signaling pathways. In the non-canonical Wnt pathway,PTK7 activates downstream signaling by direct interaction with RACK1 andrecruitment of DSH into the membrane localized receptor complex. PTK7exerts an inhibitory effect on canonical Wnt pathway signal transductionthrough competition for frizzled receptor binding at the membranesurface. PTK7 gene expression is regulated by Cdx, while proteinstability is regulated by membrane associated proteinase degradation.PTK7 is targeted for proteolytic degradation and extracellular domainshedding by the metalloproteinases MMP14 and Adam17, leading to enhancedcell proliferation, migration, and facilitated cancer cell invasion(Peradziryi, et al. Arch Biochem Biophys. 524, 2012). Soluble PTK7(sPTK7) was used to show a role for PTK7 in VEGF-induced angiogenesis,as well as, in vitro tube formation, migration and invasion of humanendothelial cells.

Within cancerous tissues, in addition to its potential for modulatingthe Wnt pathways, PTK appears to convey pro-proliferation andanti-apoptotic signals in the HCT116 colon carcinoma line, phenotypeswhich could be reversed by RNAi mediated knock-down of PTK7 (Meng, etal., 2010, PLoS One 5(11):e14018). PTK7 anti-apoptotic signals conveyedresistance to anthracycline-mediated cell killing in adult myeloidleukemia (AML) blasts, which could be reversed using a soluble PTK7-Fcprotein (Prebet, et al., 2010, Blood 116(13):2315-23). Overexpression ofPTK7 by specific cancer cells has been exploited in a strategy to targetdelivery of daunorubicin to T-ALL cells in culture using aptamers thatbind PTK7 and are subsequently internalized.

II. PTK7 Antibody-Drug Conjugates

The present invention provides antibody-drug conjugates of the formulaAb-(L-D), wherein (a) Ab is an antibody, or antigen-binding fragmentthereof, that binds to PTK7, and (b) L-D is a linker-drug moiety,wherein L is a linker, and D is a drug. Also provided are methods ofpreparing and manufacturing such antibody-drug conjugates, and use ofthe same in clinical applications. “Antibody-drug conjugate” or “ADC”refers to antibodies, or antigen-binding fragments thereof, includingantibody derivatives that bind to PTK7 and are conjugated to a drug suchas a cytotoxic, cytostatic, and/or therapeutic agent, as describedfurther herein below. For example, a cytotoxic agent can be linked orconjugated to an anti-PTK7 antibody as described herein for targetedlocal delivery of the cytotoxic agent to tumors (e.g., PTK7 expressingtumors).

As used herein, the term “PTK7” includes variants, isoforms, homologs,orthologs and paralogs. PTK7 is also known in the art as colon carcinomakinase 4 (CCK4 or CCK-4). For the purposes of the instant application itwill be appreciated that the terms “PTK7” and “CCK4” are usedinterchangeably and include splice variants, isoforms, species orthologsand homologs of human PTK7 or human CCK4. It will further be appreciatedthat the terms may also refer to any derivative or fragment of a nativeor variant form of PTK7 or CCK4 containing an epitope to which a PTK7antibody can specifically bind.

In some aspects of the invention, antibodies and antibody-drugconjugates cross-react with PTK7 from species other than human, such asPTK7 of mouse, rat, or primate, as well as different forms of PTK7(e.g., glycosylated PTK7). In other aspects, the antibodies andantibody-drug conjugates may be completely specific for human PTK7 andmay not exhibit species or other types of cross-reactivity. As usedherein the term PTK7 refers to naturally occurring human PTK7 unlesscontextually dictated otherwise. Therefore, a “PTK7 antibody” or“anti-PTK7 antibody” or other similar designation, means any antibody(as defined herein) that associates, binds or reacts with human PTK7, orfragment or derivative thereof. Further, a “PTK7 antibody-drugconjugate” or “anti-PTK7 antibody-drug conjugate” means anyantibody-drug conjugate or ADC (as defined herein) that associates,binds or reacts with human PTK7, or fragment or derivative thereof.

“Linker (L)” describes the direct or indirect linkage of the antibody tothe drug. Attachment of a linker to an antibody can be accomplished in avariety of ways, such as through surface lysines, reductive-coupling tooxidized carbohydrates, cysteine residues liberated by reducinginterchain disulfide linkages, reactive cysteine residues engineered atspecific sites, and acyl donor glutamine-containing tag or an endogenousglutamine made reactive by polypeptide engineering in the presence oftransglutaminase and an amine. A variety of ADC linkage systems areknown in the art, including hydrazone-, disulfide- and peptide-basedlinkages.

“Drug (D)” is any substance having biological or detectable activity,for example, therapeutic agents, detectable labels, binding agents,etc., and prodrugs, which are metabolized to an active agent in vivo.The terms drug, payload, and compound are used interchangeably.

“L-D” is a linker-drug moiety resulting from a drug (D) linked to alinker (L).

Additional scientific and technical terms used in connection with thepresent invention, unless indicated otherwise herein, shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclature used in connection with, and techniques of, celland tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art.

In a particular aspect of the invention, a PTK7 antibody-drug conjugateof the formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain variable region set forthas SEQ ID NO: 1 and a light chain variable region set forth as SEQ IDNO: 15; and (b) a linker-drug moiety, L-D, wherein L is a linker, and Dis a drug, wherein the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:13 and a light chain set forth as SEQ ID NO: 23; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain variable region set forthas SEQ ID NO: 25 and a light chain variable region set forth as SEQ IDNO: 39; and (b) a linker-drug moiety, L-D, wherein L is a linker, and Dis a drug, wherein the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:37 and a light chain set forth as SEQ ID NO: 47; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain variable region set forthas SEQ ID NO: 49 and a light chain variable region set forth as SEQ IDNO: 63; and (b) a linker-drug moiety, L-D, wherein L is a linker, and Dis a drug, wherein the linker ismaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

In another aspect of the invention, a PTK7 antibody-drug conjugate ofthe formula Ab-(L-D) comprises (a) an antibody, or antigen-bindingfragment thereof, Ab, comprising a heavy chain set forth as SEQ ID NO:61 and a light chain set forth as SEQ ID NO: 71; and (b) a linker-drugmoiety, L-D, wherein L is a linker, and D is a drug, wherein the linkeris maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc), andwherein the drug is 0101.

The DAR (drug-to-antibody ratio) or drug loading, indicating the numberof drug molecules conjugated per antibody, may be from 1 to 8.Compositions, batches, and/or formulations of a plurality ofantibody-drug conjugates may be characterized by an average DAR. DAR andaverage DAR can be determined by various conventional means such as UVspectroscopy, mass spectroscopy, ELISA assay, radiometric methods,hydrophobic interaction chromatography (HIC), electrophoresis and HPLC.

In particular aspects of the invention, purified anti-PTK7 ADCs may haveno unconjugated antibodies (free antibodies) present. In other aspectsof the invention, the purified anti-PTK7 ADCs may be monomeric ADCs, andthe aggregates and dimers are absent. In other aspects of the invention,the purified anti-PTK7 ADCs may have no free drug present. In furtheraspects of the invention, the purified anti-PTK7 ADCs may be monomericADCs and have no free drug present.

IIA. PTK7 Antibodies

For preparation of PTK7 antibody-drug conjugates of the invention, theantibody, or antigen-binding fragment thereof, can be any antibody, orantigen-binding fragment thereof, that specifically binds to PTK7. Theantibodies the present invention are further disclosed and characterizedin PCT International Publication No. WO 2012/112943, which isincorporated herein by reference in its entirety. More particularly, thesequences of PTK7 antibodies disclosed therein, including complete heavyand light chains, variable regions thereof, and CDRs thereof, areincorporated herein by reference. For use in preparation of PTK7antibody-drug conjugates, the antibody, or antigen-binding fragmentthereof, may be isolated, purified, or derivatized.

An “antibody” or “Ab” is an immunoglobulin molecule capable ofrecognizing and binding to a specific target or antigen, such as acarbohydrate, polynucleotide, lipid, polypeptide, etc., through at leastone antigen recognition site, located in the variable region of theimmunoglobulin molecule. As used herein, the term “antibody” canencompass any type of antibody, including but not limited to monoclonalantibodies, polyclonal antibodies, “antigen-binding fragments” (orportion), such as Fab, Fab′, F(ab′)₂, Fd, Fv, Fc, etc., of intactantibodies that retain the ability to specifically bind to a givenantigen (e.g. PTK7), an isolated complementarity determining region(CDR), bispecific antibodies, heteroconjugate antibodies, mutantsthereof, fusion proteins having an antibody, or antigen-binding fragmentthereof, (e.g., a domain antibody), single chain (ScFv) and singledomain antibodies (e.g., shark and camelid antibodies), maxibodies,minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR andbis-scFv (see, e.g., Holliger and Hudson, 2005, Nature Biotechnology23(9): 1126-1136), humanized antibodies, chimeric antibodies and anyother modified configuration of the immunoglobulin molecule thatincludes an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. Theantibodies may be murine, rat, human, or any other origin (includingchimeric or humanized antibodies). In some aspects of the invention, theantibody, or antigen-binding fragment thereof, of the disclosed PTK7antibody-drug conjugates is a chimeric, humanized, or a recombinanthuman antibody, or PTK7-binding fragment thereof.

An antibody, an antibody-drug conjugate, or a polypeptide that“specifically binds” or “preferentially binds” (used interchangeablyherein) to a target or antigen (e.g., PTK7 protein) is a term wellunderstood in the art, and methods to determine such specific orpreferential binding are also well known in the art. A molecule is saidto exhibit “specific binding” or “preferential binding” if it reacts orassociates more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular cell or substance than it doeswith alternative cells or substances. An antibody “specifically binds”or “preferentially binds” to a target or antigen if it binds withgreater affinity, avidity, more readily, and/or with greater durationthan it binds to other substances. For example, an antibody thatspecifically or preferentially binds to a PTK7 epitope is an antibodythat binds this epitope with greater affinity, avidity, more readily,and/or with greater duration than it binds to other PTK7 epitopes ornon-PTK7 epitopes.

The term “binding affinity” or “K_(D)” as used herein, is intended torefer to the equilibrium dissociation constant of a particularantigen-antibody interaction. The K_(D) is the ratio of the rate ofdissociation, also called the “off-rate” or “K_(d)”, to the rate ofassociation, or “on-rate” or “K_(a)”. Thus, K_(D) equals K_(d)/K_(a) andis expressed as a molar concentration (M). It follows that the smallerthe K_(D), the stronger the binding affinity. Therefore, a K_(D) of 1 μMindicates weak binding affinity compared to a K_(D) of 1 nM. K_(D)values for antibodies can be determined using methods well establishedin the art. One method for determining the K_(D) of an antibody is byusing surface plasmon resonance, typically using a biosensor system suchas a BIACORE® system.

Specific binding of the disclosed PTK7 antibody-drug conjugates refersto a preferential binding of an antibody-drug conjugate to human PTK7antigen in a heterogeneous sample having multiple different antigens.Typically, specific binding occurs if the binding affinity of theantibody-drug conjugate, or antibody portion (Ab) thereof, is at leastabout 10⁻⁷ M or higher, such as at least about 10⁻⁸ M or higher,including at least about 10⁻⁹ M or higher, at least about 10⁻¹⁰ M orhigher, at least about 10⁻¹¹ M or higher, or at least about 10⁻¹² M orhigher. For example, specific binding of an antibody-drug conjugate, orantibody portion (Ab) thereof, of the invention to a human PTK7 antigenincludes binding in the range of at least about 1×10⁻⁷ M to about1×10⁻¹² M, such as within the range of about 1×10⁻⁸ M to about 1×10⁻¹²M, or within the range of about 1×10⁻⁸ M to about 1×10⁻¹¹ M, or withinthe range of about 1×10⁻⁸ M to about 1×10⁻¹⁰ M, or within the range ofabout 1×10⁻⁹ M to about 1×10⁻¹⁰ M. Specific binding also refers toselective targeting of a PTK7 antibody-drug conjugate, or antibodyportion (Ab) thereof, to PTK7-expressing cells following administrationof the antibody to a subject.

It is also understood that an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

As used herein, “epitope” includes any protein determinant capable ofspecific binding to an immunoglobulin or T-cell receptor or otherwiseinteracting with a molecule. Epitopic determinants generally consist ofchemically active surface groupings of molecules such as amino acids orcarbohydrate or sugar side chains and generally have specific threedimensional structural characteristics, as well as specific chargecharacteristics. An epitope may be ‘linear’ or ‘conformational.’ In alinear epitope, all of the points of interaction between the protein andthe interacting molecule (such as an antibody) occur linearly along theprimary amino acid sequence of the protein. In a conformational epitope,the points of interaction include amino acid residues on the proteinthat are separated from one another. Once a desired epitope on anantigen is determined, it is possible to generate antibodies to thatepitope, e.g., using the techniques described in the present invention.Alternatively, during the discovery process, the generation andcharacterization of antibodies may elucidate information about desirableepitopes. From this information, it is then possible to competitivelyscreen antibodies for binding to the same epitope. An approach toachieve this is to conduct cross-competition studies to find antibodiesthat competitively bind with one another, i.e. the antibodies competefor binding to the antigen. A high throughput process for ‘binning’antibodies based upon their cross-competition is described in PCTInternational Publication No. WO 03/48731. As used herein, the term‘binning’ refers to a method to group antibodies based on their antigenbinding characteristics and competition with each other.

An “isolated antibody”, as used herein, refers to an antibody that issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that specifically binds PTK7is substantially free of antibodies that specifically bind antigensother than PTK7). Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. An isolated antibodythat specifically binds PTK7 may, however, have cross-reactivity toother antigens, such as PTK7 molecules from other species (i.e. anortholog) or with more than one isoform of PTK7.

In some aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody that competes for binding to human PTK7 with,and/or binds the same epitope as, an antibody, or antigen-bindingfragment thereof, having (a) a heavy chain variable region set forth asSEQ ID NO: 1 and a light chain variable region set forth as SEQ ID NO:15; or (c) a heavy chain variable region set forth as SEQ ID NO: 25 anda light chain variable region set forth as SEQ ID NO: 39; or (c) a heavychain variable region set forth as SEQ ID NO: 49 and a light chainvariable region set forth as SEQ ID NO: 63.

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen-binding fragment thereof, binds toan epitope in a manner sufficiently similar to the binding of a secondantibody, or an antigen-binding fragment thereof, such that the resultof binding of the first antibody with its cognate epitope is detectablydecreased in the presence of the second antibody compared to the bindingof the first antibody in the absence of the second antibody. Thealternative, where the binding of the second antibody to its epitope isalso detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Bothcompeting and cross-competing antibodies are encompassed by the presentinvention. Regardless of the mechanism by which such competition orcross-competition occurs (e.g., steric hindrance, conformational change,or binding to a common epitope, or portion thereof), the skilled artisanwould appreciate, based upon the teachings provided herein, that suchcompeting and/or cross-competing antibodies are encompassed and can beuseful for the methods disclosed herein.

Native or naturally occurring antibodies, and native immunoglobulins,are typically heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. Each light chain is linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies among theheavy chains of different immunoglobulin isotypes. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at one end a variable domain (VH) followed by a number ofconstant domains. Each light chain has a variable domain at one end (VL)and a constant domain at its other end; the constant domain of the lightchain is aligned with the first constant domain of the heavy chain, andthe light chain variable domain is aligned with the variable domain ofthe heavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains. The term“variable” refers to the fact that certain portions of the variabledomains differ extensively in sequence among antibodies.

Antibodies and the above-noted antibody domains may be described as“polypeptides”, “oligopeptides”, “peptides” and “proteins”, i.e., chainsof amino acids of any length, preferably, relatively short (e.g., 10-100amino acids). The chain may be linear or branched, it may comprisemodified amino acids, and/or may be interrupted by non-amino acids. Itis further understood that the polypeptides can occur as single chainsor associated chains. Amino acids may be referred to herein by eithertheir commonly known three letter symbols or by the one-letter symbolsrecommended by the IUPAC-IUB Biochemical. The terms “polypeptides”,“oligopeptides”, “peptides” and “proteins” also encompass an amino acidchain that has been modified naturally or by intervention; for example,disulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification, such asconjugation with a labeling component. Also included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids, etc.),as well as other modifications known in the art. Amino acidmodifications can be made by any method known in the art and many suchmethods are well known and routine for the skilled artisan. For example,but not by way of limitation, amino acid substitutions, deletions andinsertions may be accomplished using any well-known PCR-based technique.Amino acid substitutions may be made by site-directed mutagenesis (see,for example, Zoller and Smith, 1982, Nucl. Acids Res. 10:6487-6500; andKunkel, 1985, Proc. Natl. Acad. Sci USA 82:488).

A “constant region” of an antibody refers to the constant region of theantibody light chain or the constant region of the antibody heavy chain,either alone or in combination. The constant regions of chimeric andhumanized PTK7 antibodies may be derived from constant regions of anyone of IgA, IgD, IgE, IgG, IgM, any isotypes thereof (e.g., IgG1, IgG2,IgG3, or IgG4 isotypes of IgG), as well as subclasses and mutatedversions thereof. Depending on the antibody amino acid sequence of theconstant region of its heavy chains, immunoglobulins can be assigned todifferent classes. There are five major classes of immunoglobulins: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Theheavy chain constant regions that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions, such as Fc receptor(FcR) binding, participation of the antibody in antibody-dependentcellular toxicity, opsonization, initiation of complement dependentcytotoxicity, and mast cell degranulation. As known in the art, the term“Fc region” is used to define a C-terminal region of an immunoglobulinheavy chain. The “Fc region” may be a native sequence Fc region or avariant Fc region. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. Thenumbering of the residues in the Fc region is that of the EU index as inKabat. Kabat et al., Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health, Bethesda,Md., 1991. The Fc region of an immunoglobulin generally having twoconstant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods,4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995.“FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol., 117:587-593 (1976); and Kim et al., European J. Immunol.,24:2429-2434 (1994)).

It has been previously reported that certain residues presumably presenton the surface of the CH2 or CH3 domain of the heavy chain ofantibodies, or on the constant domain of the light chain, or otherwiseaccessible, are suitable for the substitution of the naturally-occurringwild type amino acid with, for example, cysteine, and are thereforeuseful to engineer a site capable of conjugation to various agents.

By “engineered Fc polypeptide”, “engineered Fc region” and “engineeredFc” as the terms are interchangeably used herein, is meant as an Fcpolypeptide, or portion thereof, comprising at least one mutation, e.g.,an amino acid substitution, introducing a site for conjugation. Themutation introduces a cysteine in place of the naturally-occurring aminoacid residue at that position, where the mutation creates a reactivesite (e.g., a reactive sulfhydryl group) for conjugation of a moiety tothe Fc.

The term “acyl donor glutamine-containing tag” or “glutamine tag” asused herein refers to a polypeptide or a protein containing one or moreGin residue(s) that acts as a transglutaminase amine acceptor.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-Lazikaniet al., J. Molec. Biol. 273:927-948 (1997). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

A CDR of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the accumulation of both Kabat and Chothia, VBASE2, AbM,contact, and/or conformational definitions or any method of CDRdetermination well known in the art. Antibody CDRs may be identified asthe hypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, (1989). The CDR positions may also be derived from ananalysis of the VBASE2 database. (See, e.g. Retter et al., 2005, NucleicAcids Res. 33(Database Issue):D671-D674).

Other approaches to CDR identification include the “AbM definition,”which is a compromise between Kabat and Chothia and is derived usingOxford Molecular's AbM antibody modeling software (now ACCELRYS®), orthe “contact definition” of CDRs based on observed antigen contacts, setforth in MacCallum et al., J. Mol. Biol., 262:732-745, (1996). Inanother approach, referred to herein as the “conformational definition”of CDRs, the positions of the CDRs may be identified as the residuesthat make enthalpic contributions to antigen binding. See, e.g., Makabeet al., Journal of Biological Chemistry, 283:1156-1166, 2008. Stillother CDR boundary definitions may not strictly follow one of the aboveapproaches, but will nonetheless overlap with at least a portion of theKabat CDRs, although they may be shortened or lengthened in light ofprediction or experimental findings that particular residues or groupsof residues or even entire CDRs do not significantly impact antigenbinding. As used herein, a CDR may refer to CDRs defined by any approachknown in the art, including combinations of approaches. The methods usedherein may utilize CDRs defined according to any of these approaches.For PTK7 antibody-drug conjugates described herein, CDRs may be definedin accordance with any of Kabat, Chothia, extended, VBASE2, AbM,contact, and/or conformational definitions.

In other aspects of the invention, the PTK7 antibody, or antigen-bindingfragment thereof, includes one or more CDR(s) of the antibody (such asone, two, three, four, five, or all six CDRs).

For the instant invention, the CDRs of hu23, hu24, and hu58 set forth inTable 1 below (SEQ ID NOS: 1-72) were derived using Kabat and Chothia.The CDRs as set forth in FIG. 6 of PCT International Publication No. WO2012/112943 which is incorporated by references herein, were derivedfrom an analysis of the VBASE2 database. Accordingly, antibodies havingCDRs defined by all such nomenclature are expressly included within thescope of the instant invention. More broadly, the term “variable regionCDR amino acid residue” includes amino acids in a CDR as identifiedusing any sequence or structure based method as set forth above.

In some aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having CDRsof a hu23 antibody. For example, a PTK7 antibody-drug conjugate mayinclude an antibody, or antigen-binding fragment thereof, including atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionhas three CDRs set forth as SEQ ID NOs: 3, 7, and 11. In some aspects ofthe invention, a PTK7 antibody-drug conjugate includes an antibody, orantigen-binding fragment thereof, having at least one heavy chainvariable region and at least one light chain variable region, whereinthe at least one light chain variable region has three CDRs set forth asSEQ ID NOs: 17, 19, and 21. A PTK7 antibody-drug conjugate of theinvention can also include an antibody, or antigen-binding fragmentthereof, including (a) a heavy chain variable region having three CDRsset forth as SEQ ID NOs: 3, 7, and 11; and (b) a light chain variableregion having three CDRs set forth as SEQ ID NOs: 17, 19, and 21.

In still other aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having one ormore hu23 CDRs defined according to Chothia or derived from an analysisof the VBASE2 database. For example, a PTK7 antibody-drug conjugate caninclude an antibody, or antigen-binding fragment thereof, having atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionincludes three hu23 CDRs defined by Chothia (see Table 1) or three hu23CDRs derived from an analysis of the VBASE2 database (see PCTInternational Publication No. WO 2012/112943). As another example, aPTK7 antibody-drug conjugate can include an antibody, or antigen-bindingfragment thereof, having at least one heavy chain variable region and atleast one light chain variable region, wherein the at least one lightchain variable region includes three hu23 CDRs defined by Chothia (seeTable 1) or three hu23 CDRs derived from an analysis of the VBASE2database (see PCT International Publication No. WO 2012/112943). In someaspects of the invention, a PTK7 antibody-drug conjugate of theinvention can include an antibody, or antigen-binding fragment thereof,having (a) a heavy chain variable region having three hu23 CDRs definedaccording to Chothia (see Table 1); and (b) a light chain variableregion having three hu23 CDRs defined according to Chothia (see Table1). In some aspects of the invention, a PTK7 antibody-drug conjugate ofthe invention can include an antibody, or antigen-binding fragmentthereof, having (a) a heavy chain variable region including three hu23CDRs derived from an analysis of the VBASE2 database (see PCTInternational Publication No. WO 2012/112943); and (b) a light chainvariable region including three hu23 CDRs derived from an analysis ofthe VBASE2 database (see PCT International Publication No. WO2012/112943).

In other aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having CDRsof a hu24 antibody. For example, a PTK7 antibody-drug conjugate mayinclude an antibody, or antigen-binding fragment thereof, having atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionincludes three CDRs set forth as SEQ ID NOs: 27, 31, and 35. In someaspects of the invention, a PTK7 antibody-drug conjugate includes anantibody, or antigen-binding fragment thereof, having at least one heavychain variable region and at least one light chain variable region,wherein the at least one light chain variable region includes three CDRsset forth as SEQ ID NOs: 41, 43, and 45. A PTK7 antibody-drug conjugateof the invention can also include (a) a heavy chain variable regionhaving three CDRs set forth as SEQ ID NOs: 27, 31, and 35; and (b) alight chain variable region having three CDRs set forth as SEQ ID NOs:41, 43, and 45.

In other aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having one ormore hu24 CDRs defined according to Chothia or derived from an analysisof the VBASE2 database. For example, a PTK7 antibody-drug conjugate caninclude an antibody, or antigen-binding fragment thereof, having atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionincludes three hu24 CDRs defined by Chothia (see Table 1) or three hu24CDRs derived from an analysis of the VBASE2 database (see PCTInternational Publication No. WO 2012/112943). As another example, aPTK7 antibody-drug conjugate can include an antibody, or antigen-bindingfragment thereof, having at least one heavy chain variable region and atleast one light chain variable region, wherein the at least one lightchain variable region includes three hu24 CDRs defined by Chothia (seeTable 1) or three hu24 CDRs derived from an analysis of the VBASE2database (see PCT International Publication No. WO 2012/112943). In someaspects of the invention, a PTK7 antibody-drug conjugate of theinvention can include an antibody, or antigen-binding fragment thereof,having (a) a heavy chain variable region having three hu24 CDRs definedaccording to Chothia (see Table 1); and (b) a light chain variableregion having three hu24 CDRs defined according to Chothia (see Table1). In some aspects of the invention, a PTK7 antibody-drug conjugate ofthe invention can include an antibody, or antigen-binding fragmentthereof, having (a) a heavy chain variable region including three hu24CDRs derived from an analysis of the VBASE2 database (see PCTInternational Publication No. WO 2012/112943); and (b) a light chainvariable region including three hu24 CDRs derived from an analysis ofthe VBASE2 database (see PCT International Publication No. WO2012/112943).

In other aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having CDRsof a hu58 antibody. For example, a PTK7 antibody-drug conjugate mayinclude an antibody, or antigen-binding fragment thereof, having atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionincludes three CDRs set forth as SEQ ID NOs: 51, 55, and 59. In someaspects of the invention, a PTK7 antibody-drug conjugate includes anantibody, or antigen-binding fragment thereof, having at least one heavychain variable region and at least one light chain variable region,wherein the at least one light chain variable region includes three CDRsset forth as SEQ ID NOs: 65, 67, and 69. A PTK7 antibody-drug conjugateof the invention can also include (a) a heavy chain variable regionhaving three CDRs set forth as SEQ ID NOs: 51, 55, and 59; and (b) alight chain variable region having three CDRs set forth as SEQ ID NOs:65, 67, and 69.

In other aspects of the invention, a PTK7 antibody-drug conjugateincludes an antibody, or antigen-binding fragment thereof, having one ormore hu58 CDRs defined according to Chothia or derived from an analysisof the VBASE2 database. For example, a PTK7 antibody-drug conjugate caninclude an antibody, or antigen-binding fragment thereof, having atleast one heavy chain variable region and at least one light chainvariable region, wherein the at least one heavy chain variable regionincludes three hu58 CDRs defined by Chothia or three hu58 CDRs derivedfrom an analysis of the VBASE2 database. As another example, a PTK7antibody-drug conjugate can include an antibody, or antigen-bindingfragment thereof, having at least one heavy chain variable region and atleast one light chain variable region, wherein the at least one lightchain variable region includes three hu58 CDRs defined by Chothia orthree hu58 CDRs derived from an analysis of the VBASE2 database. In someaspects of the invention, a PTK7 antibody-drug conjugate of theinvention can include an antibody, or antigen-binding fragment thereof,having (a) a heavy chain variable region having three hu58 CDRs definedaccording to Chothia; and (b) a light chain variable region having threehu58 CDRs defined according to Chothia. In some aspects of theinvention, a PTK7 antibody-drug conjugate of the invention can includean antibody, or antigen-binding fragment thereof, having (a) a heavychain variable region including three hu58 CDRs derived from an analysisof the VBASE2 database; and (b) a light chain variable region includingthree hu24 CDRs derived from an analysis of the VBASE2 database.

In some aspects of the invention, antibodies used to prepare PTK7antibody-drug conjugates of the invention will be monoclonal antibodies.The term “monoclonal antibody” or “mAb” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present invention may be made by the hybridomamethod first described by Kohler and Milstein, Nature 256:495-497(1975), or may be made by recombinant DNA methods such as described inU.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., Nature 348:552-554 (1990) for example.

In some aspects of the invention, antibodies used to prepareantibody-drug conjugates of the invention will be monovalent, i.e.,having one antigen binding site per molecule (e.g., IgG or Fab). In someinstances, a monovalent antibody can have more than one antigen bindingsites, but the binding sites are from different antigens. In someaspects of the invention, the antibody, or antigen-binding fragmentthereof, of an antibody-drug conjugate of the invention may include a“bivalent antibody”, i.e., having two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. Alternatively, bivalent antibodies may bebispecific. A “bispecific,” “dual-specific” or “bifunctional” antibodyis a hybrid antibody having two different antigen binding sites. The twoantigen binding sites of a bispecific antibody bind to two differentepitopes, which may reside on the same or different protein targets.

The term “chimeric antibody” is intended to refer to antibodies in whichpart or all of the variable region sequences are derived from onespecies and the constant region sequences are derived from anotherspecies, such as an antibody in which the variable region sequences arederived from a mouse antibody and the constant region sequences arederived from a human antibody.

As used herein, “humanized” or “CDR grafted” antibody refers to forms ofnon-human (e.g. murine) antibodies that are chimeric immunoglobulins,immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen binding subsequences of antibodies) thatcontain minimal sequence derived from a non-human immunoglobulin.Preferably, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from one or more complementary determiningregions (CDRs) of the recipient are replaced by residues from one ormore CDRs of a non-human species (donor antibody) such as mouse, rat, orrabbit having the desired specificity, affinity, and capacity.

In some instances, Fv framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may include residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences, but are included to further refine and optimize antibodyperformance. In general, the humanized antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will include at least a portion of animmunoglobulin constant region or domain (Fc), typically that of a humanimmunoglobulin. In some aspects of the invention the antibodies have Fcregions modified as described in PCT International Publication No. WO99/58572. Other forms of humanized antibodies have one or more CDRs (CDRL1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) which may be altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody.

Humanization can be essentially performed following the method of Winterand co-workers (Jones et al. Nature 321:522-525 (1986); Riechmann et al.Nature 332:323-327 (1988); Verhoeyen et al. Science 239:1534-1536(1988)), by substituting rodent or mutant rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. See also U.S. Pat.Nos. 5,225,539; 5,585,089; 5,693,761; 5,693,762; 5,859,205; which areincorporated herein by reference in its entirety. In some instances,residues within the framework regions of one or more variable regions ofthe human immunoglobulin are replaced by corresponding non-humanresidues (see, for example, U.S. Pat. Nos. 5,585,089; 5,693,761;5,693,762; and 6,180,370). Furthermore, humanized antibodies may includeresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance (e.g., to obtain desired affinity). In general, thehumanized antibody will include substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will include at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details see Jones et al. Nature 321:522-525 (1986); Riechmann etal. Nature 332:323-327 (1988); and Presta Curr. Op. Struct. Biol.2:593-596 (1992); which are incorporated herein by reference in theirentirety. Accordingly, such “humanized” antibodies may includeantibodies wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some frameworkresidues are substituted by residues from analogous sites in rodentantibodies. See, for example, U.S. Pat. Nos. 5,225,539; 5,585,089;5,693,761; 5,693,762; 5,859,205. See also U.S. Pat. No. 6,180,370, andPCT International Publication No. WO 01/27160, where humanizedantibodies and techniques for producing humanized antibodies havingimproved affinity for a predetermined antigen are disclosed.

“Recombinant human antibody” or “fully human antibody” refers to thoseantibodies having an amino acid sequence corresponding to that of anantibody produced by a human and/or which has been made using any of thetechniques for making human antibodies known to those skilled in the artor disclosed herein. This definition of a human antibody includesantibodies having at least one human heavy chain polypeptide or at leastone human light chain polypeptide. One such example is an antibodyhaving murine light chain and human heavy chain polypeptides. Humanantibodies can be produced using various techniques known in the art.For example, a human antibody is selected from a phage library, wherethat phage library expresses human antibodies (Vaughan et al., NatureBiotechnology, 14:309-314, (1996); Sheets et al., Proc. Natl. Acad. Sci.(USA) 95:6157-6162, (1998); Hoogenboom and Winter, J. Mol. Biol.,227(2):381-388 (1992); Marks et al., J. Mol. Biol., 222(3):581-597(1991)). Human antibodies can also be made by immunization of animalsinto which human immunoglobulin loci have been transgenically introducedin place of the endogenous loci, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Thisapproach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibodymay be prepared by immortalizing human B lymphocytes that produce anantibody directed against a target antigen (such B lymphocytes may berecovered from an individual or from single cell cloning of the cDNA, ormay have been immunized in vitro). See, e.g., Cole et al. MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, p. 77, (1985); Boerner etal., J. Immunol., 147 (1):86-95, (1991); and U.S. Pat. No. 5,750,373.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50 (1999) and Fellouse, F. A., et al, J. Mol.Biol., 373(4):924-40 (2007)). Additional guidance may be found inSambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(2000); Ausubel et al., Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1998); and Coligan et al., Short Protocols in ProteinScience, Wiley, John & Sons, Inc. (2003). Representative methods arealso described in Example 1 herein below.

In general, for the production of hybridoma cell lines, the route andschedule of immunization of the host animal are generally in keepingwith established and conventional techniques for antibody stimulationand production. It is contemplated that any mammalian subject includinghumans or antibody producing cells therefrom can be manipulated to serveas the basis for production of mammalian, including human and hybridomacell lines. Typically, the host animal is inoculated intraperitoneally,intramuscularly, orally, subcutaneously, intraplantar, and/orintradermally with an amount of immunogen, including as describedherein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C., Nature 256:495-497, 1975 or as modified by Buck, D.W., et al., In Vitro, 18:377-381, 1982. Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce the PTK7 monoclonal antibodies of the subjectinvention. The hybridomas are expanded and subcloned, if desired, andsupernatants are assayed for anti-immunogen activity by conventionalimmunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, orfluorescence immunoassay). Hybridomas that may be used as source ofantibodies encompass all derivatives, progeny cells of the parenthybridomas that produce monoclonal antibodies specific for PTK7, or aportion thereof.

Hybridomas that produce such antibodies may be grown in vitro or in vivousing known procedures. The monoclonal antibodies may be isolated fromthe culture media or body fluids, by conventional immunoglobulinpurification procedures such as ammonium sulfate precipitation, gelelectrophoresis, dialysis, chromatography, and ultrafiltration, ifdesired. Undesired activity, if present, can be removed, for example, byrunning the preparation over adsorbents made of the immunogen attachedto a solid phase and eluting or releasing the desired antibodies off theimmunogen. Immunization of a host animal with a human PTK7, or afragment containing the target amino acid sequence conjugated to aprotein that is immunogenic in the species to be immunized, e.g.,keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, the PTK7 antibody (monoclonal or polyclonal) of interest maybe sequenced and the polynucleotide sequence may then be cloned into avector for expression or propagation. The sequence encoding the antibodyof interest may be maintained in vector in a host cell and the host cellcan then be expanded and frozen for future use. Production ofrecombinant monoclonal antibodies in cell culture can be carried outthrough cloning of antibody genes from B cells by means known in theart. See, e.g. Tiller et al., J. Immunol. Methods 329:112-124 (2008);U.S. Pat. No. 7,314,622.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

The term “vector” means a construct, which is capable of delivering,and, preferably, expressing, one or more gene(s) or sequence(s) ofinterest in a host cell. Examples of vectors include, but are notlimited to, viral vectors, naked DNA or RNA expression vectors, plasmid,cosmid or phage vectors, DNA or RNA expression vectors associated withcationic condensing agents, DNA or RNA expression vectors encapsulatedin liposomes, and certain eukaryotic cells, such as producer cells.

The term “expression control sequence” means a nucleic acid sequencethat directs transcription of a nucleic acid. An expression controlsequence can be a promoter, such as a constitutive or an induciblepromoter, or an enhancer. The expression control sequence is operablylinked to the nucleic acid sequence to be transcribed.

Alternatively, the polynucleotide sequence may be used for geneticmanipulation to “humanize” the antibody or to improve the affinity, orother characteristics of the antibody. For example, the constant regionmay be engineered to more nearly resemble human constant regions toavoid immune response if the antibody is used in clinical trials andtreatments in humans. It may be desirable to genetically manipulate theantibody sequence to obtain greater affinity to PTK7 and greaterefficacy in inhibiting PTK7.

There are four general steps that may be used to humanize a monoclonalantibody: (1) determining the nucleotide and predicted amino acidsequence of the starting antibody light and heavy variable domains (2)designing the humanized antibody, i.e., deciding which antibodyframework region to use during the humanizing process (3) the actualhumanizing methodologies/techniques and (4) the transfection andexpression of the humanized antibody. See, for example, U.S. Pat. Nos.4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761;5,693,762; 5,585,089; and 6,180,370.

Humanized antibodies may be prepared using any one of a variety ofmethods including veneering, grafting of complementarity determiningregions (CDRs), grafting of abbreviated CDRs, grafting of specificitydetermining regions (SDRs), and Frankenstein assembly, as describedbelow. Humanized antibodies also include superhumanized antibodies, inwhich one or more changes have been introduced in the CDRs. For example,human residues may be substituted for non-human residues in the CDRs.These general approaches may be combined with standard mutagenesis andsynthesis techniques to produce an anti-PTK7 antibody of any desiredsequence.

Veneering is based on the concept of reducing potentially immunogenicamino acid sequences in a rodent or other non-human antibody byresurfacing the solvent accessible exterior of the antibody with humanamino acid sequences. Thus, veneered antibodies appear less foreign tohuman cells than the unmodified non-human antibody. See Padlan (1991)Mol. Immunol. 28:489-98. A non-human antibody is veneered by identifyingexposed exterior framework region residues in the non-human antibody,which are different from those at the same positions in frameworkregions of a human antibody, and replacement of the identified residueswith amino acids that typically occupy these same positions in humanantibodies.

Grafting of CDRs is performed by replacing one or more CDRs of anacceptor antibody (e.g., a human antibody or other antibody havingdesired framework residues) with CDRs of a donor antibody (e.g., anon-human antibody). Acceptor antibodies may be selected based onsimilarity of framework residues between a candidate acceptor antibodyand a donor antibody. For example, according to the Frankensteinapproach, human framework regions are identified as having substantialsequence homology to each framework region of the relevant non-humanantibody, and CDRs of the non-human antibody are grafted onto thecomposite of the different human framework regions. A related methodalso useful for preparation of antibodies of the invention is describedin U.S. Pat. No. 7,321,026.

Grafting of abbreviated CDRs is a related approach. Abbreviated CDRsinclude the specificity-determining residues and adjacent amino acids,including those at positions 27d-34, 50-55 and 89-96 in the light chain,and at positions 31-35b, 50-58, and 95-101 in the heavy chain (numberingconvention of (Kabat et al. (1987)). See (Padlan et al. (1995) FASEB J.9: 133-9). Grafting of specificity-determining residues (SDRs) ispremised on the understanding that the binding specificity and affinityof an antibody combining site is determined by the most highly variableresidues within each of the complementarity determining regions (CDRs).Analysis of the three-dimensional structures of antibody-antigencomplexes, combined with analysis of the available amino acid sequencedata may be used to model sequence variability based on structuraldissimilarity of amino acid residues that occur at each position withinthe CDR. SDRs are identified as minimally immunogenic polypeptidesequences consisting of contact residues. See Padlan et al. (1995) FASEBJ. 9: 133-139.

In general, human acceptor frameworks are selected on the basis thatthey are substantially similar to the framework regions of the donorantibodies, or which are most similar to the consensus sequence of thevariable region subfamily. Following grafting, additional changes may bemade in the donor and/or acceptor sequences to optimize antibodybinding, functionality, codon usage, expression levels, etc., includingintroduction of non-human residues into the framework regions. See e.g.,PCT International Publication No. WO 91/09967.

For grafting of CDRs onto a heavy chain variable framework region,useful framework sequences may be derived from a DP-21 (VH7), DP-54(VH3-07), DP-47 (VH3-23), DP-53 (VH-74), DP-49 (VH3-30), DP-48 (VH3-13),DP-75, DP-8(VH1-2), DP-25, VI-2b and VI-3 (VH1-03), DP-15 and V1-8(VH1-08), DP-14 and V1-18 (VH1-18), DP-5 and V1-24P (VH1-24), DP-4(VH1-45), DP-7 (VH1-46), DP-10, DA-6 and YAC-7 (VH1-69), DP-88 (VH1-e),DP-3 and DA-8 (VH1-f). For grafting of CDRs onto a light chain variableframework region, useful framework sequences may be derived from a DPK24subgroup IV germ line clone, a Will subgroup (DPK23, DPK22, DPK20,DPK21), or a VκI subgroup germ line clone (DPK9, DPK1, O2, DPK7).

Antigen-binding fragments or antibody fragments can be produced byproteolytic or other degradation of the antibodies, by recombinantmethods (i.e., single or fusion polypeptides) as described above or bychemical synthesis. Polypeptides of the antibodies, especially shorterpolypeptides up to about 50 amino acids, are conveniently made bychemical synthesis. Methods of chemical synthesis are known in the artand are commercially available. For example, an antibody or antibodyfragment could be produced by an automated polypeptide synthesizeremploying the solid phase method. See also, U.S. Pat. Nos. 5,807,715;4,816,567; and 6,331,415.

In other aspects of the invention, the PTK7 antibody-drug conjugatesinclude an antibody, or antigen-binding fragment thereof, having a hu23,hu24, or hu58 heavy chain and/or light chain variable region, or avariable region substantially similar to a hu23, hu24, or hu58 heavychain or light chain variable region.

As applied to polypeptides, the term “substantial identity” or“substantial similarity” means that two amino acid sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights as supplied with the programs, share at least 70%, 75% or80% sequence identity, preferably at least 90% or 95% sequence identity,and more preferably at least 97%, 98% or 99% sequence identity. In somesubstantially similar amino acid sequences, residue positions that arenot identical differ by conservative amino acid substitutions.

Substantially similar polypeptides also include conservativelysubstituted variants in which one or more residues have beenconservatively substituted with a functionally similar residue. Examplesof conservative substitutions include the substitution of one non-polar(hydrophobic) residue such as isoleucine, valine, leucine or methioninefor another; the substitution of one polar (hydrophilic) residue foranother such as between arginine and lysine, between glutamine andasparagine, between glycine and serine; the substitution of one basicresidue such as lysine, arginine or histidine for another; or thesubstitution of one acidic residue, such as aspartic acid or glutamicacid for another.

A further indication that two proteins are substantially identical isthat they share an overall three-dimensional structure, or arebiologically functional equivalents.

In some aspects of the invention, an antibody-drug conjugate, whichbinds to PTK7, includes an antibody, or antigen-binding fragmentthereof, having a heavy chain variable region set forth as any one ofSEQ ID NOs: 1, 25, or 49 and/or a light chain variable region set forthas any one of SEQ ID NOs: 15, 39, or 63. For example, a PTK7antibody-drug conjugate of the invention can include an antibody, orantigen-binding fragment thereof, having a heavy chain variable regionhaving an amino acid sequence that is at least 90% identical to SEQ IDNO: 1 and a light chain variable region having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 15; or an antibody, orantigen-binding fragment thereof, having a heavy chain variable regionset forth as SEQ ID NO: 1 and a light chain variable region having anamino acid sequence set forth as SEQ ID NO: 15. As another example, aPTK7 antibody-drug conjugate of the invention can include an antibody,or antigen-binding fragment thereof, having a heavy chain variableregion having an amino acid sequence that is at least 90% identical toSEQ ID NO: 25 and a light chain variable region having an amino acidsequence that is at least 90% identical to SEQ ID NO: 39; or anantibody, or antigen-binding fragment thereof, having a heavy chainvariable region set forth as SEQ ID NO: 25 and a light chain variableregion having an amino acid sequence set forth as SEQ ID NO: 39. Asanother example, a PTK7 antibody-drug conjugate of the invention caninclude an antibody, or antigen-binding fragment thereof, having a heavychain variable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 49 and a light chain variable region having anamino acid sequence that is at least 90% identical to SEQ ID NO: 63; oran antibody, or antigen-binding fragment thereof, having a heavy chainvariable region set forth as SEQ ID NO: 49 and a light chain variableregion having an amino acid sequence set forth as SEQ ID NO: 63.

The antibodies may also be modified, e.g. in the variable domains of theheavy and/or light chains, e.g., to alter a binding property of theantibody. For example, a mutation may be made in one or more of the CDRregions to increase or decrease the K_(D) of the antibody for PTK7, toincrease or decrease K_(off), or to alter the binding specificity of theantibody. Techniques in site-directed mutagenesis are well-known in theart. See, e.g., Sambrook et al. and Ausubel et al., supra.

A modification or mutation may also be made in a framework region orconstant region to increase the half-life of a PTK7 antibody. See, e.g.PCT International Publication No. WO 00/09560. A mutation in a frameworkregion or constant region can also be made to alter the immunogenicityof the antibody, to provide a site for covalent or non-covalent bindingto another molecule, or to alter such properties as complement fixation,FcR binding and antibody-dependent cell-mediated cytotoxicity. Accordingto the invention, a single antibody may have mutations in any one ormore of the CDRs or framework regions of the variable domain or in theconstant region.

In a process known as “germlining”, certain amino acids in the VH and VLsequences can be mutated to match those found naturally in germline VHand VL sequences. In particular, the amino acid sequences of theframework regions in the VH and VL sequences can be mutated to match thegermline sequences to reduce the risk of immunogenicity when theantibody is administered. As used herein, the term “germline” refers tothe nucleotide sequences and amino acid sequences of the antibody genesand gene segments as they are passed from parents to offspring via thegerm cells. This germline sequence is distinguished from the nucleotidesequences encoding antibodies in mature B cells which have been alteredby recombination and hypermutation events during the course of B cellmaturation. An antibody that “utilizes” a particular germline has anucleotide or amino acid sequence that most closely aligns with thatgermline nucleotide sequence or with the amino acid sequence that itspecifies. Such antibodies frequently are mutated compared with thegermline sequence. Germline DNA sequences for human VH and VL genes areknown in the art (see e.g., the “Vbase” human germline sequencedatabase; see also Kabat, E. A., et al., 1991, Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson et al., J. Mol.Biol. 227:776-798, 1992; and Cox et al., Eur. J. Immunol. 24:827-836,1994.

Another type of amino acid substitution that may be made is to removepotential proteolytic sites in the antibody. Such sites may occur in aCDR or framework region of a variable domain or in the constant regionof an antibody. Substitution of cysteine residues and removal ofproteolytic sites may decrease the risk of heterogeneity in the antibodyproduct and thus increase its homogeneity. Another type of amino acidsubstitution is to eliminate asparagine-glycine pairs, which formpotential deamidation sites, by altering one or both of the residues. Inanother example, the C-terminal lysine of the heavy chain of a PTK7antibody of the invention can be cleaved. In various aspects of theinvention, the heavy and light chains of the PTK7 antibodies mayoptionally include a signal sequence.

To express the PTK7 antibodies of the present invention, DNA fragmentsencoding VH and VL regions can first be obtained using any of themethods described above. As known in the art, “polynucleotide,” “nucleicacid/nucleotide,” and “oligonucleotide” are used interchangeably herein,and include polymeric forms of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, analogs thereof, or anysubstrate that can be incorporated into a chain by DNA or RNApolymerase. Polynucleotides may have any three-dimensional structure,and may perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, DNA, cDNA, genomic DNA, recombinant polynucleotides, branchedpolynucleotides, plasmids, vectors, isolated DNA of any sequence,isolated RNA of any sequence, nucleic acid probes, and primers.Polynucleotides may be naturally-occurring, synthetic, recombinant orany combination thereof. A polynucleotide may include modifiednucleotides, such as methylated nucleotides and their analogs. Ifpresent, modification to the nucleotide structure may be imparted beforeor after assembly of the chain. The sequence of nucleotides may beinterrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications include, for example,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha- or beta-anomeric sugars, epimeric sugars such as arabinose,xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses,acyclic analogs and abasic nucleoside analogs such as methyl riboside.One or more phosphodiester linkages may be replaced by alternativelinking groups. These alternative linking groups include, but are notlimited to, features wherein phosphate is replaced by P(O)S(“thioate”),P(S)S (“dithioate”), (O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

Representative DNAs encoding anti-PTK7 antibody heavy chain and lightchain variable regions are set forth as SEQ ID NO: 2 (hu23 VH DNA), SEQID NO: 16 (hu23 VL DNA), SEQ ID NO: 26 (hu24 VH DNA), SEQ ID NO: 40(hu24 VL DNA), SEQ ID NO: 50 (hu58 VH DNA) and SEQ ID NO: 64 (hu58 VLDNA). Representative DNAs encoding anti-PTK7 antibody heavy chains andlight chains are set forth as SEQ ID NO: 14 (hu23 HC DNA), SEQ ID NO: 24(hu23 LC DNA), SEQ ID NO: 38 (hu24 HC DNA), SEQ ID NO: 48 (hu24 LC DNA),SEQ ID NO: 62 (hu58 HC DNA), and SEQ ID NO: 72 (hu58 LC DNA)

Various modifications, e.g. mutations, substitutions, deletions, and/oradditions can also be introduced into the hu23, hu24, and hu58 DNAsequences using standard methods known to those of skill in the art. Forexample, mutagenesis can be carried out using standard methods, such asPCR-mediated mutagenesis, in which the mutated nucleotides areincorporated into the PCR primers such that the PCR product contains thedesired mutations or site-directed mutagenesis.

Accordingly, based upon the disclosure of the instant application, oneskilled in the art would readily recognize the sequences of DNAssubstantially similar hu23, hu24, and hu58 DNAs. The term “substantialsimilarity” or “substantial sequence similarity,” when referring to anucleic acid or fragment thereof, means that when optimally aligned withappropriate nucleotide insertions or deletions with another nucleic acid(or its complementary strand), there is nucleotide sequence identity inat least about 85%, preferably at least about 90%, and more preferablyat least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, asmeasured by any well-known algorithm of sequence identity, such asFASTA, BLAST or Gap.

The term “percent sequence identity” in the context of nucleic acidsequences means the residues in two sequences that are the same whenaligned for maximum correspondence. The length of sequence identitycomparison may be over a stretch of at least about nine nucleotides,usually at least about 18 nucleotides, more usually at least about 24nucleotides, typically at least about 28 nucleotides, more typically atleast about 32 nucleotides, and preferably at least about 36, 48 or morenucleotides. There are a number of different algorithms known in the artwhich can be used to measure nucleotide sequence identity. For instance,polynucleotide sequences can be compared using FASTA, Gap or Bestfit,which are programs in Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis. FASTA, which includes, e.g., the programsFASTA2 and FASTA3, provides alignments and percent sequence identity ofthe regions of the best overlap between the query and search sequences(Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol.132:185-219 (2000); Pearson, Methods Enzymol. 266:227-258 (1996);Pearson, J. Mol. Biol. 276:71-84 (1998); which are incorporated hereinby reference in its entirety). Unless otherwise specified, defaultparameters for a particular program or algorithm are used. For instance,percent sequence identity between nucleic acid sequences can bedetermined using FASTA with its default parameters (a word size of 6 andthe NOPAM factor for the scoring matrix) or using Gap with its defaultparameters as provided in GCG Version 6.1, which is incorporated hereinby reference in its entirety.

A further indication that two nucleic acid sequences are substantiallyidentical is that proteins encoded by the nucleic acids aresubstantially identical, share an overall three-dimensional structure,or are biologically functional equivalents. These terms are definedfurther herein below. Nucleic acid molecules that do not hybridize toeach other under stringent conditions are still substantially identicalif the corresponding proteins are substantially identical. This mayoccur, for example, when two nucleotide sequences compriseconservatively substituted variants as permitted by the genetic code.

Conservatively substituted variants are nucleic acid sequences havingdegenerate codon substitutions wherein the third position of one or moreselected (or all) codons are substituted with mixed-base and/ordeoxyinosine residues. See Batzer et al. (1991) Nucleic Acids Res.19:5081; Ohtsuka et al. (1985) J. Biol. Chem. 260:2605-2608; andRossolini et al. (1994) Mol. Cell Probes 8:91-98.

One type of substitution, for example, that may be made is to change oneor more cysteines in the antibody, which may be chemically reactive, toanother residue, such as, without limitation, alanine or serine. Forexample, there can be a substitution of a non-canonical cysteine. Thesubstitution can be made in a CDR or framework region of a variabledomain or in the constant region of an antibody. As another example, thecysteine may be canonical.

Once DNA fragments encoding the VH and VL segments of the presentinvention are obtained, these DNA fragments can be further manipulatedby standard recombinant DNA techniques, for example to convert thevariable region genes to full-length antibody chain genes, to Fabfragment genes, or to a scFv gene. In these manipulations, a VL- orVH-encoding DNA fragment is operatively linked to another DNA fragmentencoding another protein, such as an antibody constant region or aflexible linker. The term “operatively linked”, as used in this context,is intended to mean that the two DNA fragments are joined such that theamino acid sequences encoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH1, CH2and CH3). The sequences of human heavy chain constant region genes areknown in the art (see e.g., Kabat, E. A., et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG2 constant region. The IgG constant region sequence can beany of the various alleles or allotypes known to occur among differentindividuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). These allotypesrepresent naturally occurring amino acid substitution in the IgG1constant regions. For a Fab fragment heavy chain gene, the VH-encodingDNA can be operatively linked to another DNA molecule encoding only theheavy chain CH1 constant region. The CH1 heavy chain constant region maybe derived from any of the heavy chain genes.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (see e.g., Kabat, E. A., etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region. The kappa constant region maybe any of the various alleles known to occur among differentindividuals, such as Inv(1), Inv(2), and Inv(3). The lambda constantregion may be derived from any of the three lambda genes.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker suchthat the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (See e.g., Bird et al., 1988, Science 242:423-426; Huston et al.,1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990,Nature 348:552-554. The single chain antibody may be monovalent, if onlya single VH and VL are used, bivalent, if two VH and VL are used, orpolyvalent, if more than two VH and VL are used. Bispecific orpolyvalent antibodies may be generated that bind specifically to PTK7and to another molecule.

In another aspect of the invention, a fusion antibody or immunoadhesinmay be made that includes all or a portion of a PTK7 antibody of theinvention linked to another polypeptide. In another aspect, only thevariable domains of the PTK7 antibody are linked to the polypeptide. Inanother aspect, the VH domain of a PTK7 antibody is linked to a firstpolypeptide, while the VL domain of a PTK7 antibody is linked to asecond polypeptide that associates with the first polypeptide in amanner such that the VH and VL domains can interact with one another toform an antigen binding site. In another aspect, the VH domain isseparated from the VL domain by a linker such that the VH and VL domainscan interact with one another. The VH-linker-VL antibody is then linkedto the polypeptide of interest. In addition, fusion antibodies can becreated in which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

Other modified antibodies may be prepared using PTK7 antibody encodingnucleic acid molecules. For instance, “Kappa bodies” (111 et al.,Protein Eng. 10:949-57, 1997), “Minibodies” (Martin et al., EMBO J.,13:5303-9, 1994), “Diabodies” (Holliger et al., Proc. Natl. Acad. Sci.USA 90:6444-6448, 1993), or “Janusins” (Traunecker et al., EMBO J.10:3655-3659, 1991 and Traunecker et al., Int. J. Cancer (Suppl.)7:51-52, 1992) may be prepared using standard molecular biologicaltechniques following the teachings of the specification.

Bispecific antibodies or antigen binding fragments can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol.79:315-321, 1990, Kostelny et al., J. Immunol. 148:1547-1553, 1992. Inaddition, bispecific antibodies may be formed as “diabodies” or“Janusins.” In some aspects of the invention, a bispecific antibodybinds to two different epitopes of PTK7. In other aspects, modifiedantibodies described above are prepared using one or more of thevariable domains or CDR regions from the PTK7 antibodies providedherein.

For use in preparation of antibody-drug conjugates, PTK7 antibodiesdescribed herein may be substantially pure, i.e., at least 50% pure(i.e., free from contaminants), more preferably, at least 90% pure, morepreferably, at least 95% pure, yet more preferably, at least 98% pure,and most preferably, at least 99% pure.

Table 1 provides the amino acid (protein) sequences and associatednucleic acid (DNA) sequences of humanized anti-PTK7 antibodies of thepresent invention. The CDRs of hu23 VH, hu23 VL, hu24 VH, hu24 VL, hu58VH, and hu58 VL as defined by Kabat and by Chothia, are set forth asseparate sequences.

TABLE 1 Sequences of humanized anti-PTK7 antibodies. SEQ ID NO.Description Sequences  1 hu23 VHQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSNMGVGWIRQPPGKALEWLA ProteinHIWWDDDKYYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVRSN YGYAWFAYWGQGTLVTVSS 2 hu23 VH CAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAG DNAACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTAACATGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCACACATTTGGTGGGATGATGATAAGTACTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGTTCGAAGTAACTATGGTTACGCCTGGTTTGCTTACTGGGGCCAAGG GACTCTGGTCACTGTCTCTTCA 3 hu23 VH CDR1 TSNMGVG Protein-Kabat  4 hu23 VH CDR1 GFSLSTSNMProtein-Chothia  5 hu23 VH CDR1 ACTAGTAACATGGGTGTGGGC DNA-Kabat  6hu23 VH CDR1 GGGTTCTCACTCAGCACTAGTAACATG DNA-Chothia  7 hu23 VH CDR2HIWWDDDKYYSPSLKS Protein-Kabat  8 hu23 VH CDR2 WWDDD Protein-Chothia  9hu23 VH CDR2 CACATTTGGTGGGATGATGATAAGTACTACAGCCCATCTCTGAAGAGC DNA-Kabat10 hu23 VH CDR2 TGGTGGGATGATGAT DNA-Chothia 11 hu23 VH CDR3 SNYGYAWFAYProtein-Kabat and Chothia 12 hu23 VH CDR3 AGTAACTATGGTTACGCCTGGTTTGCTTACDNA-Kabat and Chothia 13 hu23 HCQITLKESGPTLVKPTQTLTLTCTFSGFSLSTSNMGVGWIRQPPGKALEWLA Protein-HuIgG1HIWWDDDKYYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCVRSNYGYAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 14 hu23 HCCAGATCACCTTGAAGGAGTCTGGTCCTACGCTGGTGAAACCCACACAG DNA-HuIgG1ACCCTCACGCTGACCTGCACCTTCTCTGGGTTCTCACTCAGCACTAGTAACATGGGTGTGGGCTGGATCCGTCAGCCCCCAGGAAAGGCCCTGGAGTGGCTTGCACACATTTGGTGGGATGATGATAAGTACTACAGCCCATCTCTGAAGAGCAGGCTCACCATCACCAAGGACACCTCCAAAAACCAGGTGGTCCTTACAATGACCAACATGGACCCTGTGGACACAGCCACATATTACTGTGTTCGAAGTAACTATGGTTACGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCGAGCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGT 15 hu23 VLDIQMTQSPSSLSASVGDRVTITCKASQDIYPYLNWFQQKPGKAPKTLIYRT ProteinNRLLDGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPLTFGAGTK LEIK 16 hu23 VLGACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAG DNAATAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTTATCCCTATTTAAACTGGTTCCAACAAAAACCAGGGAAAGCTCCTAAGACCCTGATCTATCGTACAAATAGATTGCTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGAACAGATTTTACTTTCACCATCAGCAGCCTGCAACCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATCAAA 17 hu23 VL CDR1 KASQDIYPYLN Protein-Kabatand Chothia 18 hu23 VL CDR1 AAGGCGAGTCAGGACATTTATCCCTATTTAAAC DNA-Kabatand Chothia 19 hu23 VL CDR2 RTNRLLD Protein-Kabat and Chothia 20hu23 VL CDR2 CGTACAAATAGATTGCTAGAT DNA-Kabat and Chothia 21 hu23 VL CDR3LQYDEFPLT Protein-Kabat and Chothia 22 hu23 VL CDR3CTACAGTATGATGAGTTTCCGCTCACG DNA-Kabat and Chothia 23 hu23 LCDIQMTQSPSSLSASVGDRVTITCKASQDIYPYLNWFQQKPGKAPKTLIYRT Protein-KappaNRLLDGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQYDEFPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC 24 hu23 LCGACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAG DNA-KappaATAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTTATCCCTATTTAAACTGGTTCCAACAAAAACCAGGGAAAGCTCCTAAGACCCTGATCTATCGTACAAATAGATTGCTAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGAACAGATTTTACTTTCACCATCAGCAGCCTGCAACCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATGAGTTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATCAAACGGACTGTGGCTGCACCAAGTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAG GGGAGAGTGT 25 hu24 VHQVQLVQSGPEVKKPGASVKVSCKASGYTFTDYAVHWVRQAPGKRLEWIG ProteinVISTYNDYTYNNQDFKGRVTMTRDTSASTAYMELSRLRSEDTAVYYCARG NSYFYALDYWGQGTSVTVSS26 hu24 VH CAGGTCCAGCTTGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGGC DNACTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTGACTATGCTGTGCATTGGGTGCGCCAGGCCCCCGGAAAAAGGCTTGAGTGGATTGGAGTGATCAGCACTTACAATGATTACACATACAATAACCAGGACTTCAAGGGCAGAGTCACCATGACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGACTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGGTAACTCCTACTTCTATGCTTTGGACTACTGGGGTCAAGGAA CCTCAGTCACCGTCTCCTCA27 hu24 VH CDR1 DYAVH Protein-Kabat 28 hu24 VH CDR1 GYTFTDYProtein-Chothia 29 hu24 VH CDR1 GACTATGCTGTGCAT DNA-Kabat 30hu24 VH CDR1 GGATACACCTTCACTGACTAT DNA-Chothia 31 hu24 VH CDR2VISTYNDYTYNNQDFKG Protein-Kabat 32 hu24 VH CDR2 STYNDY Protein-Chothia33 hu24 VH CDR2 GTGATCAGCACTTACAATGATTACACATACAATAACCAGGACTTCAAGGDNA-Kabat GC 34 hu24 VH CDR2 AGCACTTACAATGATTAC DNA-Chothia 35hu24 VH CDR3 GNSYFYALDY Protein-Kabat and Chothia 36 hu24 VH CDR3GGTAACTCCTACTTCTATGCTTTGGACTAC DNA-Kabat and Chothia 37 hu24 HCQVQLVQSGPEVKKPGASVKVSCKASGYTFTDYAVHWVRQAPGKRLEWIG Protein-HuIgG1VISTYNDYTYNNQDFKGRVTMTRDTSASTAYMELSRLRSEDTAVYYCARGNSYFYALDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 38 hu24 HCCAGGTCCAGCTTGTGCAGTCTGGGCCTGAGGTGAAGAAGCCTGGGGC DNA-HuIgG1CTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTGACTATGCTGTGCATTGGGTGCGCCAGGCCCCCGGAAAAAGGCTTGAGTGGATTGGAGTGATCAGCACTTACAATGATTACACATACAATAACCAGGACTTCAAGGGCAGAGTCACCATGACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGACTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGGTAACTCCTACTTCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCGAGCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGA 39 hu24 VLEIVLTQSPATLSLSPGERATLSCRASESVDSYGKSFMHWYQQKPGQAPRL ProteinLIYRASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNEDPWTF GGGTKLEIK 40hu24 VL GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG DNAAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTGACAGCTATGGCAAAAGTTTTATGCACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAGGGCATCCAACCTGGAATCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA 41 hu24 VL CDR1RASESVDSYGKSFMH Protein-Kabat and Chothia 42 hu24 VL CDR1AGGGCCAGTGAGAGTGTTGACAGCTATGGCAAAAGTTTTATGCAC DNA-Kabat and Chothia 43hu24 VL CDR2 RASNLES Protein-Kabat and Chothia 44 hu24 VL CDR2AGGGCATCCAACCTGGAATCT DNA-Kabat and Chothia 45 hu24 VL CDR3 QQSNEDPWTProtein-Kabat and Chothia 46 hu24 VL CDR3 CAGCAGAGTAATGAGGATCCGTGGACGDNA-Kabat and Chothia 47 hu24 LCEIVLTQSPATLSLSPGERATLSCRASESVDSYGKSFMHWYQQKPGQAPRL Protein-KappaLIYRASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSNEDPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 48hu24 LC GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGG DNA-KappaAAAGAGCCACCCTCTCCTGCAGGGCCAGTGAGAGTGTTGACAGCTATGGCAAAAGTTTTATGCACTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATAGGGCATCCAACCTGGAATCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGACTGTGGCTGCACCAAGTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT 49 hu58 VHEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYVVMSWVRQAPGKGLEWI ProteinGDLNPDSSAINYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTLITTLVPYTMDFWGQGTSVTVSS 50 hu58 VHGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG DNAGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCGACTTTAGTAGATATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGCGACCTAAACCCAGATTCAAGTGCGATAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACACTCATTACTACGTTAGTACCCTATACTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA 51 hu58 VH CDR1 RYWMS Protein-Kabat 52hu58 VH CDR1 GFDFSRY Protein-Chothia 53 hu58 VH CDR1 AGATATTGGATGAGCDNA-Kabat 54 hu58 VH CDR1 GGATTCGACTTTAGTAGATAT DNA-Chothia 55hu58 VH CDR2 DLNPDSSAINYVDSVKG Protein-Kabat 56 hu58 VH CDR2 NPDSSAProtein-Chothia 57 hu58 VH CDR2GACCTAAACCCAGATTCAAGTGCGATAAACTATGTGGACTCTGTGAAGG DNA-Kabat GC 58hu58 VH CDR2 AACCCAGATTCAAGTGCG DNA-Chothia 59 hu58 VH CDR3 ITTLVPYTMDFProtein-Kabat and Chothia 60 hu58 VH CDR3ATTACTACGTTAGTACCCTATACTATGGACTTC DNA-Kabat and Chothia 61 hu58 HCEVQLVESGGGLVQPGGSLRLSCAASGFDFSRYWMSWVRQAPGKGLEWI Protein-HuIgG1GDLNPDSSAINYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTLITTLVPYTMDFWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 62 hu58 HCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGG DNA-HuIgG1GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCGACTTTAGTAGATATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGATCGGCGACCTAAACCCAGATTCAAGTGCGATAAACTATGTGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACACTCATTACTACGTTAGTACCCTATACTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCGAGCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGT 63 hu58 VLETTLTQSPAFMSATPGDKVNISCITNTDIDDDMNWYQQKPGEAAILLISEGN ProteinGLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGSGTKL EIK 64 hu58 VLGAAACGACACTCACGCAGTCTCCAGCATTCATGTCAGCGACTCCAGGA DNAGACAAAGTCAACATCTCCTGCATAACCAACACAGACATTGATGATGATATGAACTGGTACCAACAGAAACCAGGAGAAGCTGCTATTCTCCTTATTTCAGAAGGTAATGGTCTCCGTCCTGGAATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCTACAAAGTGATAACTTGCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA 65 hu58 VL CDR1 ITNTDIDDDMN Protein-Kabatand Chothia 66 hu58 VL CDR1 ATAACCAACACAGACATTGATGATGATATGAAC DNA-Kabatand Chothia 67 hu58 VL CDR2 EGNGLRP Protein-Kabat and Chothia 68hu58 VL CDR2 GAAGGTAATGGTCTCCGTCCT DNA-Kabat and Chothia 69 hu58 VL CDR3LQSDNLPLT Protein-Kabat and Chothia 70 hu58 VL CDR3CTACAAAGTGATAACTTGCCTCTCACG DNA-Kabat and Chothia 71 hu58 LCETTLTQSPAFMSATPGDKVNISCITNTDIDDDMNWYQQKPGEAAILLISEGN Protein-KappaGLRPGIPPRFSGSGYGTDFTLTINNIESEDAAYYFCLQSDNLPLTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC 72 hu58 LCGAAACGACACTCACGCAGTCTCCAGCATTCATGTCAGCGACTCCAGGA DNA-KappaGACAAAGTCAACATCTCCTGCATAACCAACACAGACATTGATGATGATATGAACTGGTACCAACAGAAACCAGGAGAAGCTGCTATTCTCCTTATTTCAGAAGGTAATGGTCTCCGTCCTGGAATCCCACCTCGATTCAGTGGCAGCGGGTATGGAACAGATTTTACCCTCACAATTAATAACATAGAATCTGAGGATGCTGCATATTACTTCTGTCTACAAAGTGATAACTTGCCTCTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAACGGACTGTGGCTGCACCAAGTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT

II.B. Linkers

Anti-PTK7 antibody-drug conjugates of the present invention can beprepared using a linker to link or conjugate a drug to an anti-PTK7antibody. A linker is a bifunctional compound which can be used to linka drug and an antibody to form an antibody drug conjugate (ADC). Suchconjugates are useful, for example, in the formation of immunoconjugatesdirected against tumor associated antigens. Such conjugates allow theselective delivery of cytotoxic drugs to tumor cells. Suitable linkersinclude, for example, cleavable and non-cleavable linkers. A cleavablelinker is typically susceptible to cleavage under intracellularconditions. Major mechanisms by which a conjugated drug is cleaved froman antibody include hydrolysis in the acidic pH of the lysosomes(hydrazones, acetals, and cis-aconitate-like amides), peptide cleavageby lysosomal enzymes (the cathepsins and other lysosomal enzymes), andreduction of disulfides. As a result of these varying mechanisms forcleavage, mechanisms of linking the drug to the antibody also varywidely and any suitable linker can be used.

Suitable cleavable linkers include, for example, a peptide linkercleavable by an intracellular protease, such as lysosomal protease or anendosomal protease. In aspects of the invention, the linker can be adipeptide linker, such as a valine-citrulline (val-cit), aphenylalanine-lysine (phe-lys) linker, ormaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc) linker.In another aspect, the linker may beSulfosuccinimidyl-4-[Nmaleimidomethyl] cyclohexane-1-carboxylate (smcc).Sulfo-smcc conjugation occurs via a maleimide group which reacts withsulfhydryls (thiols, —SH), while its Sulfo-NHS ester is reactive towardprimary amines (as found in Lysine and the protein or peptideN-terminus). Further, the linker may be maleimidocaproyl (mc).

Other suitable linkers include linkers hydrolyzable at a specific pH ora pH range, such as a hydrazone linker. Additional suitable cleavablelinkers include disulfide linkers. The linker may be covalently bound tothe antibody to such an extent that the antibody must be degradedintracellularly in order for the drug to be released e.g. the mc linkerand the like.

In particular aspects of the invention, the linker of PTK7 antibody-drugconjugates of the invention may be ormaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (vc),maleimidocaproyl (mc) or AcBut.

An example of a suitable conjugation procedure relies on the conjugationof hydrazides and other nucleophiles to the aldehydes generated byoxidation of the carbohydrates that naturally occur on antibodies.Hydrazone-containing conjugates can be made with introduced carbonylgroups that provide the desired drug-release properties. Conjugates canalso be made with a linker that has a disulfide at one end, an alkylchain in the middle, and a hydrazine derivative at the other end. Theanthracyclines are one example of cytotoxins that can be conjugated toantibodies using this technology.

Linkers containing functional groups other than hydrazones have thepotential to be cleaved in the acidic milieu of the lysosomes. Forexample, conjugates can be made from thiol-reactive linkers that containa site other than a hydrazone that is cleavable intracellularly, such asesters, amides, and acetals/ketals. Camptothecin is one cytotoxic agentthat can be conjugated using these linkers. Ketals made from a 5 to7-member ring ketone and that has one of the oxygens attached to thecytotoxic agent and the other to a linker for antibody attachment alsocan be used. The anthracyclines are also an example of a suitablecytotoxin for use with these linkers.

Another example of a class of pH sensitive linkers are thecis-aconitates, which have a carboxylic acid juxtaposed to an amidebond. The carboxylic acid accelerates amide hydrolysis in the acidiclysosomes. Linkers that achieve a similar type of hydrolysis rateacceleration with several other types of structures can also be used.The maytansinoids are an example of a cytotoxin that can be conjugatedwith linkers attached at C-9.

Another potential release method for drug conjugates is the enzymatichydrolysis of peptides by the lysosomal enzymes. In one example, apeptide is attached via an amide bond to para-aminobenzyl alcohol andthen a carbamate or carbonate is made between the benzyl alcohol and thecytotoxic agent. Cleavage of the peptide leads to the collapse, orself-immolation, of the aminobenzyl carbamate or carbonate. Thecytotoxic agents exemplified with this strategy include anthracyclines,taxanes, mitomycin C, and the auristatins. In one example, a phenol canalso be released by collapse of the linker instead of the carbamate. Inanother variation, disulfide reduction is used to initiate the collapseof a para-mercaptobenzyl carbamate or carbonate.

Many of the cytotoxic agents conjugated to antibodies have little, ifany, solubility in water and that can limit drug loading on theconjugate due to aggregation of the conjugate. One approach toovercoming this is to add solublizing groups to the linker. Conjugatesmade with a linker consisting of PEG and a dipeptide can been used,including those having a PEG di-acid, thiol-acid, or maleimide-acidattached to the antibody, a dipeptide spacer, and an amide bond to theamine of an anthracycline or a duocarmycin analogue. Another example isa conjugate prepared with a PEG-containing linker disulfide bonded to acytotoxic agent and amide bonded to an antibody. Approaches thatincorporate PEG groups may be beneficial in overcoming aggregation andlimits in drug loading.

U.S. Pat. No. 5,773,001, which is incorporated herein by reference inits entirety, discloses linkers that may be used with nucleophilicdrugs, particularly hydrazides and related nucleophiles, prepared fromthe calicheamicins. These linkers are especially useful in those caseswhere better activity is obtained when the linkage formed between thedrug and the linker is hydrolysable. These linkers contain twofunctional groups, including (1) a group for reaction with an antibody(e.g., carboxylic acid), and (2) a carbonyl group (e.g., an aldehyde ora ketone) for reaction with a drug. The carbonyl groups may react with ahydrazide group on the drug to form a hydrazone linkage. This linkage iscleavable hydrolysable, allowing for release of the therapeutic agentfrom the conjugate after binding to the target cells. In particularaspects of the invention, the linker of PTK7 antibody-drug conjugates ofthe invention may 4-(4-acetylphenoxy) butanoic acid (AcBut). In otheraspects of the invention, antibody-drug conjugates can be prepared using(3-Acetylphenyl) acetic acid (AcPAc) or 4-mercapto-4-methyl-pentanoicacid (Amide) as the linker molecule.

N-hydroxysuccinimide (OSu) esters or other comparably activated esterscan be used to generate the activated hydrolyzable linker-drug moiety.Examples of other suitable activating esters include NHS(N-hydroxysuccinimide), sulfo-NHS (sulfonated NHS), PFP(pentafluorophenyl), TFP (tetrafluorophenyl), and DNP (dinitrophenyl).

In some aspects of the invention, the antibody-drug conjugates areprepared by reacting calicheamicin or derivatives thereof, the AcButlinker and an anti-PTK7 antibody of the present invention. See e.g.,U.S. Pat. No. 5,773,001. The AcBut linker produces conjugates that aresubstantially stable in circulation, releasing an estimated 2% of thecalicheamicin per day when assayed at 37° C. in human plasma in vitro.The conjugates release the calicheamicin in the acidic lysosomes.

In some aspects of the invention, the AcButCM moiety can be generatedusing methods and processes described in the art, such as PCTInternational Publication No. WO 08/147765 and in U.S. Pat. No.8,273,862, which are incorporated herein by reference in their entirety.In some aspects of the invention, the AcButCM moiety can be generatedusing an improved synthesis process, as described in U.S. ProvisionalApplication No. 61/899,682, which is incorporated herein by reference inits entirety.

Representative linkers useful for conjugation of radioisotopes includediethylenetriamine pentaacetate (DTPA)-isothiocyanate, succinimidyl6-hydrazinium nicotinate hydrochloride (SHNH), and hexamethylpropyleneamine oxime (HMPAO) (Bakker et al. (1990) J. Nucl. Med. 31: 1501-1509,Chattopadhyay et al. (2001) Nucl. Med. Biol. 28: 741-744, Dewanjee etal. (1994) J. Nucl. Med. 35: 1054-63, Krenning et al. (1989) Lancet 1:242-244, Sagiuchi et al. (2001) Ann. Nucl. Med. 15: 267-270); U.S. Pat.No. 6,024,938). Alternatively, a targeting molecule may be derivatizedso that a radioisotope may be bound directly to it (Yoo et al. (1997) J.Nucl. Med. 38: 294-300). Iodination methods are also known in the art,and representative protocols may be found, for example, in Krenning etal. (1989) Lancet 1:242-4 and in Bakker et al. (1990) J. Nucl. Med.31:1501-9.

II.C. Drugs

Drugs useful in preparation of the disclosed PTK7 antibody-drugconjugates include any substance having biological or detectableactivity, for example, therapeutic agents, detectable labels, bindingagents, etc., and prodrugs, which are metabolized to an active agent invivo. A drug may also be a drug derivative, wherein a drug has beenfunctionalized to enable conjugation with an antibody of the invention.In accordance with the disclosed methods, the drugs are used to prepareantibody-drug conjugates of the formula Ab-(L-D), wherein (a) Ab is anantibody, or antigen-binding fragment thereof, that binds to PTK7; and(b) L-D is a linker-drug moiety, wherein L is a linker, and D is a drug.The drug-to-antibody ratio (DAR) or drug loading indicates the number ofdrug (D) molecules that are conjugated per antibody. The antibody-drugconjugates of the present invention have a DAR that is within the rangeof 1 to 8. Thus, in aspects of the invention, a PTK7 antibody-drugconjugate may include 1 drug molecule (DAR of 1), or 2 drug molecules(DAR of 2), or 3 drug molecules (DAR of 3), or 4 drug molecules (DAR of4), or 5 drug molecules (DAR of 5), or 6 drug molecules (DAR of 6), or 7drug molecules (DAR of 7), or 8 drug molecules (DAR of 8). DAR can bedetermined by various conventional means such as UV spectroscopy, massspectroscopy, ELISA assay, radiometric methods, hydrophobic interactionchromatography (HIC), electrophoresis and HPLC.

Compositions, batches and/or formulations of antibody-drug conjugate(ADC), of the formula Ab-(L-D), may include a plurality of antibodies,each antibody conjugated to a particular number of drug molecules (fromDAR 1 to 8). The compositions, batches and/or formulations have anaverage DAR.

In particular aspects of the invention, a composition, batch, and/orformulation of antibody-drug conjugates may be characterized by anaverage DAR in the range of about 1 to about 8, for example, an averageDAR in the range of about 2 to about 7, or an average DAR in the rangeof about 3 to about 6, or an average DAR in the range of about 4 toabout 5, or an average DAR in the range of about 5 to about 7, or anaverage DAR in the range of about 6 to about 8. In some aspects thecompositions, batches and/or formulations of antibody-drug conjugate mayhave an average DAR of about 1, or an average DAR of about 2, or anaverage DAR of about 3, or an average DAR of about 4, or an average DARof about 5, or an average DAR of about 6, or an average DAR of about 7,or an average DAR of about 8. As used in the foregoing ranges of averageDAR, the term “about” means+/−0.5%.

Moreover, a composition, batch, and/or formulation of antibody-drugconjugates may be characterized by a preferred range of average DAR,e.g., an average DAR in the range of about 3 to about 5, an average DARin the range of about 3 to about 4, or an average DAR in the range ofabout 4 to about 5. Further, a composition, batch, and/or formulation ofantibody-drug conjugates may be characterized by a preferred range ofaverage DAR, e.g., an average DAR in the range of 3 to 5, an average DARin the range of 3 to 4, or an average DAR in the range of 4 to 5.

Compositions, batches and/or formulations of ADCs of the formulaAb-(L-D), may be characterized by a DAR distribution. The DARdistribution provides the percent or fraction of various ADC species,e.g. DAR 1 to 8 that may be present in a composition, batch, and/orformulation of ADCs. The DAR distribution of a composition, batch,and/or formulation of ADCs may be determined by methods known in theart, such as capillary iso-electric focusing (cIEF).

In one aspect of the invention, the DAR distribution of a composition,batch, and/or formulation of ADCs, of the formula Ab-(L-D), may becharacterized as a highly heterogeneous mixture of ADCs with a broad DARdistribution, generally containing a broad range of ADC species with DAR1 to 8.

In another aspect of the invention, the DAR distribution of acomposition, batch, and/or formulation of ADCs may be characterized as ahighly homogeneous mixture with a narrow DAR distribution, generallycontaining a narrow range of ADC species having a particular DAR, suchas DAR 3 to 5.

For example, a therapeutic agent is an agent that exerts a cytotoxic,cytostatic, and/or immunomodulatory effect on cancer cells or activatedimmune cells. Examples of therapeutic agents include cytotoxic agents,chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.Chemotherapeutic agents are chemical compounds useful in the treatmentof cancer.

Therapeutic agents are compositions that may be used to treat or preventa disorder in a subject in need thereof. Therapeutic agents useful inthe invention include anti-cancer agents, i.e., agents havinganti-cancer activity in PTK7-expressing cells such as cancer cells frombreast cancer, such as triple-negative breast cancer (TNBC),progesterone-receptor positive breast cancer (PR+), estrogen-receptorpositive breast cancer (ER+) and double positive breast cancer; ovariancancer; colorectal cancer; leukemias, such as acute myeloid leukemia(AML) and acute lymphoblastic leukemia (ALL); esophageal cancer; gastriccancer; melanoma; sarcoma; kidney cancer; pancreatic cancer; prostatecancer; liver cancer, such as hepatocellular carcinoma (HCC); and lungcancer, such as non-small cell lung cancer (NSCLC) and small cell lungcancer (SCLC).

Representative therapeutic agents include cytotoxins, cytotoxic agents,and cytostatic agents. A cytotoxic effect refers to the depletion,elimination and/or the killing of a target cell(s). A cytotoxic agentrefers to an agent that has a cytotoxic and/or cytostatic effect on acell. A cytostatic effect refers to the inhibition of cellproliferation. A cytostatic agent refers to an agent that has acytostatic effect on a cell, thereby inhibiting the growth and/orexpansion of a specific subset of cells.

Additional representative therapeutic agents include radioisotopes,chemotherapeutic agents, immunomodulatory agents, anti-angiogenicagents, anti-proliferative agents, pro-apoptotic agents, and cytolyticenzymes (e.g., RNAses). An agent may also include a therapeutic nucleicacid, such as a gene encoding an immunomodulatory agent, ananti-angiogenic agent, an anti-proliferative agent, or a pro-apoptoticagent. These drug descriptors are not mutually exclusive, and thus atherapeutic agent may be described using one or more of the above-notedterms. For example, selected radioisotopes are also cytotoxins.Therapeutic agents may be prepared as pharmaceutically acceptable salts,acids or derivatives of any of the above. Generally, conjugates having aradioisotope as the drug are referred to as radioimmunoconjugates andthose having a chemotherapeutic agent as the drug are referred to aschemoimmunoconjugates.

Examples of a cytotoxic agents include, but are not limited to ananthracycline, an auristatin, CC-1065, a dolastatin, a duocarmycin, anenediyne, a geldanamycin, a maytansine, a puromycin, a taxane, a vincaalkaloid, a SN-38, a tubulysin, a hemiasterlin, and stereoisomers,isosteres, analogs or derivatives thereof. Chemotherapeutic agents,plant toxins, other bioactive proteins, enzymes (i.e., ADEPT),radioisotopes, photosensitizers (i.e., for photodynamic therapy) canalso be used. In one embodiment, the cytotoxic agent is not a ribosomeinactiviating protein. In a more specific embodiment, the cytotoic agentis not saporin.

The anthracyclines are derived from bacteria Strepomyces and have beenused to treat a wide range of cancers, such as leukemias, lymphomas,breast, uterine, ovarian, and lung cancers. Exemplary anthracyclinesinclude, but are not limited to, daunorubicin, doxorubicin (i.e.,adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

Dolastatins and their peptidic analogs and derivatives, auristatins, arehighly potent antimitotic agents that have been shown to have anticancerand antifungal activity. See, e.g., U.S. Pat. No. 5,663,149 and Pettitet al., Antimicrob. Agents Chemother. 42:2961-2965, (1998). Exemplarydolastatins and auristatins include, but are not limited to, dolastatin10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD(Monomethyl Auristatin D or monomethyl dolastatin 10), MMAF (MonomethylAuristatin F orN-methylvaline-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE(Monomethyl Auristatin E orN-methylvaline-valine-dolaisoleuine-dolaproine-norephedrine),5-benzoylvaleric acid-AE ester (AEVB).

In some aspects of the invention, auristatins described in PCTInternational Publication No. WO 2013/072813, which is incorporatedherein by reference in its entirety, and methods of producing thoseauristatins are used herein.

For example, the auristatin is 0101,(2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),having the following structure:

In another example, the auristatin is 8261, (82612-Methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide),having the following structure:

Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxicpotency. See Boger and Johnson, PNAS 92:3642-3649, 1995. Exemplarydolastatins and auristatins include, but are not limited to,(+)-docarmycin A and (+)-duocarmycin SA, and (+)-CC-1065.

Enediynes are a class of anti-tumor bacterial products characterized byeither nine- and ten-membered rings or the presence of a cyclic systemof conjugated triple-double-triple bonds. Exemplary enediynes include,but are not limited to, calicheamicin, esperamicin, and dynemicin.

In some aspects of the invention, the cytotoxic agent is an antibiotic,such as calicheamicin, also called the LL-E33288 complex, for example,β-calicheamicin, γ-calicheamicin or N-acetyl-γ-calicheamicin(gamma-calicheamicin (γ₁)). Examples of calicheamicins suitable for usein the present invention are disclosed, for example, in U.S. Pat. Nos.4,671,958 4,970,198, 5,053,394, 5,037,651, 5,079,233 and 5,108,912,which are incorporated herein by reference in its entirety. Thesecompounds contain a methyltrisulfide that may be reacted withappropriate thiols to form disulfides, at the same time introducing afunctional group such as a hydrazide or other functional group that isuseful for conjugating calicheamicin to an anti-PTK7 antibody. Disulfideanalogs of calicheamicin can also be used, for example, analogsdescribed in U.S. Pat. Nos. 5,606,040 and 5,770,710, which areincorporated herein by reference in its entirety. In some aspects of theinvention, the disulfide analog is N-acetyl-γ-calicheamicin dimethylhydrazide (hereinafter “CM”).

Geldanamycins are benzoquinone ansamycin antibiotic that bind to Hsp90(Heat Shock Protein 90) and have been used antitumor drugs. Exemplarygeldanamycins include, but are not limited to, 17-AAG(17-N-Allylamino-17-Demethoxygeldanamycin) and 17-DMAG(17-Dimethylaminoethylamino-17-demethoxygeldanamycin).

Maytansines or their derivatives maytansinoids inhibit cellproliferation by inhibiting the microtubules formation during mitosisthrough inhibition of polymerization of tubulin. See Remillard et al.,Science 189:1002-1005, 1975. Exemplary maytansines and maytansinoidsinclude, but are not limited to, mertansine (DM1) and its derivatives aswell as ansamitocin.

Taxanes are diterpenes that act as anti-tubulin agents or mitoticinhibitors. Exemplary taxanes include, but are not limited to,paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

Vinca alkyloids are also anti-tubulin agents. Exemplary vinca alkyloidsinclude, but are not limited to, vincristine, vinblastine, vindesine,and vinorelbine.

In some aspects of the invention, the agent is an immunomodulatingagent. Examples of an immunomodulating agent include, but are notlimited to, gancyclovier, etanercept, tacrolimus, sirolimus,voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine,mycophenolgate mofetil, methotrextrate, glucocorticoid and its analogs,cytokines, xanthines, stem cell growth factors, lymphotoxins, tumornecrosis factor (TNF), hematopoietic factors, interleukins (e.g.,interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21),colony stimulating factors (e.g., granulocyte-colony stimulating factor(G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)),interferons (e.g., interferons-α, -β and -γ), the stem cell growthfactor designated “S 1 factor,” erythropoietin and thrombopoietin, or acombination thereof.

Immunomodulatory agents useful in the invention also includeanti-hormones that block hormone action on tumors and immunosuppressiveagents that suppress cytokine production, down-regulate self-antigenexpression, or mask MHC antigens. Representative anti-hormones includeanti-estrogens including, for example, tamoxifen, raloxifene, aromataseinhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene,LY 117018, onapnstone, and toremifene; and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andanti-adrenal agents. Representative immunosuppressive agents include2-amino-6-aryl-5-substituted pyrimidines, azathioprine,cyclophosphamide, bromocryptine, danazol, dapsone, glutaraldehyde,anti-idiotypic antibodies for MHC antigens and MHC fragments,cyclosporin A, steroids such as glucocorticosteroids, cytokine orcytokine receptor antagonists (e.g., anti-interferon antibodies,anti-IL10 antibodies, anti-TNFα antibodies, anti-IL2 antibodies),streptokinase, TGFβ, rapamycin, T-cell receptor, T-cell receptorfragments, and T cell receptor antibodies.

In some aspects of the invention, the drug is a therapeutic proteinincluding, but is not limited to, a toxin, a hormone, an enzyme, and agrowth factor.

Examples of a toxin protein (or polypeptide) include, but are notlimited to, dipththeria (e.g., diphtheria A chain), Pseudomonas exotoxinand endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain),modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, ribonuclease (RNase), DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),momordica charantia inhibitor, curcin, crotin, sapaonaria officinalisinhibitor, mitogellin, restrictocin, phenomycin, enomycin,tricothecenes, inhibitor cystine knot (ICK) peptides (e.g.,ceratotoxins), and conotoxin (e.g., KIIIA or SmIIIa).

Examples of hormones include, but are not limited to, estrogens,androgens, progestins and corticosteroids.

In some aspects of the invention, the cytotoxic agent can be made usinga liposome or biocompatible polymer. The anti-PTK7 antibodies asdescribed herein can be conjugated to the biocompatible polymer toincrease serum half-life and bioactivity, and/or to extend in vivohalf-lives. Examples of biocompatible polymers include water-solublepolymer, such as polyethylene glycol (PEG) or its derivatives thereofand zwitterion-containing biocompatible polymers (e.g., aphosphorylcholine containing polymer).

In some aspects of the invention, the drug is an oligonucleotide, suchas anti-sense oligonucleotides.

Additional drugs useful in the invention include anti-angiogenic agentsthat inhibit blood vessel formation, for example, farnesyltransferaseinhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroidsulphatase inhibitors (e.g., 2-methoxyoestradiol bis-sulphamate(2-MeOE2bisMATE)), interleukin-24, thrombospondin, metallospondinproteins, class I interferons, interleukin 12, protamine, angiostatin,laminin, endostatin, and prolactin fragments.

Anti-proliferative agents and pro-apoptotic agents include activators ofPPAR-gamma (e.g., cyclopentenone prostaglandins (cyPGs)), retinoids,triterpinoids (e.g., cycloartane, lupane, ursane, oleanane, friedelane,dammarane, cucurbitacin, and limonoid triterpenoids), inhibitors of EGFreceptor (e.g., HER4), rampamycin, CALCITRIOL®(1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors(FEMARA® (letrozone)), telomerase inhibitors, iron chelators (e.g.,3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin(viral protein 3—VP3 from chicken aneamia virus), inhibitors of Bcl-2and Bcl-X(L), TNF-alpha, FAS ligand, TNF-related apoptosis-inducingligand (TRAIL/Apo2L), activators of TNF-alpha/FAS ligand/TNF-relatedapoptosis-inducing ligand (TRAIL/Apo2L) signaling, and inhibitors ofPI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).

Representative chemotherapeutic agents include alkylating agents such asthiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan,improsulfan and piposulfan; aziidines such as benzodopa, carboquone,meturedopa, and uredopa; ethylenimines and methylamelamines includingaltretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylolomelamine; nitrogen mustardssuch as chlorambucil, chlornaphazine, cholophosphamide, estramustine,ifosfamide, mechiorethamine, mechiorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfarnide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, ranimustine; antibiotics such asaclacinomysins, actinomycin, authramycin, azaserine, bleomycins,cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin,chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-EU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asarninoglutethimide, mitotane, trilostane; folic acid replenishers suchas frolinic acid; aceglatone; aldophospharnide glycoside;arninolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate;defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate;etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine;mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet;pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine;razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2′-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology of Princeton, N.J.)and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer of Antony, France);chiorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aininopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; andcapecitabine.

Additional therapeutic agents that may be used in accordance with thepresent invention include photosensitizing agents, such as U.S. Pat.Nos. 7,498,029 and 5,952,329, which are incorporated herein by referencein its entirety, for photodynamic therapy; magnetic particles forthermotherapy, such as U.S. Pat. No. 6,997,863, which is incorporatedherein by reference in its entirety; binding agents, such as peptides,ligands, cell adhesion ligands, etc., and prodrugs such asphosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate containing prodrugs, peptide containing prodrugs,β-lactam-containing prodrugs, substituted phenoxyacetamide-containingprodrugs or substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs that may beconverted to the more active cytotoxic free drug.

For diagnostic methods using anti-PTK7 antibodies, a drug may include adetectable label used to detect the presence of PTK7-expressing cells invitro or in vivo. Radioisotopes that are detectable in vivo, such asthose labels that are detectable using scintigraphy, magnetic resonanceimaging, or ultrasound, may be used in clinical diagnostic applications.Useful scintigraphic labels include positron emitters and γ-emitters.Representative contrast agents for magnetic source imaging areparamagnetic or superparamagnetic ions (e.g., iron, copper, manganese,chromium, erbium, europium, dysprosium, holmium and gadolinium), ironoxide particles, and water soluble contrast agents. For ultrasonicdetection, gases or liquids may be entrapped in porous inorganicparticles that are released as microbubble contrast agents. For in vitrodetection, useful detectable labels include fluorophores, detectableepitopes or binding agents, and radioactive labels.

Thus, in some aspects of the invention, the drug is an imaging agent(e.g., a fluorophore or a PET (Positron Emission Tomography) label,SPECT (Single-Photon Emission Computed Tomorgraphy) label), or MRI(Magnetic Resonance Imaging) label.

The term “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody soas to generate a “labeled” antibody. The label may be detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable. Radionuclides that can serveas detectable labels include, for example, 1-131, 1-123, 1-125, Y-90,Re-188, Re-186, At-211, Cu-67, Bi-212, and Pd-109. The label might alsobe a non-detectable entity such as a toxin.

Examples of fluorophores include, but are not limited to, fluoresceinisothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g.,5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g.,Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633,647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g.,5,-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g.,SR101).

Therapeutic or diagnostic radioisotopes or other labels (e.g., PET orSPECT labels) can be incorporated in the agent for conjugation to theanti-PTK7 antibodies as described herein. The isotope may be directlybound to the antibody, for example, at a cysteine residue present in theantibody, or a chelator may be used to mediate the binding of theantibody and the radioisotope. Radioisotopes suitable for radiotherapyinclude but are not limited to α-emitters, β-emitters, and augerelectrons. For diagnostic applications, useful radioisotopes includepositron emitters and γ-emitters. An anti-PTK7 antibody of the inventionmay further be iodinated, for example, on a tyrosine residue of theantibody, to facilitate detection or therapeutic effect of the antibody.

Examples of a radioisotope or other labels include, but are not limitedto, ³H, ¹¹C, ¹³N, ¹⁴C, ¹⁵N, ¹⁵O, ³⁵S, ¹⁸F, ³²P, ³³P, ⁴⁷Sc, ⁵¹Cr, ⁵⁷Co,⁵⁸Co, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁵Se, ⁷⁶Br, ⁷⁷Br, ⁸⁶Y, ⁸⁹Zr,⁹⁰Y, ⁹⁴Tc, ⁹⁵Ru, ⁹⁷Ru, ⁹⁹Tc, ¹⁰³Ru, ¹⁰⁵Rh, ¹⁰⁵Ru, ¹⁰⁷Hg, ¹⁰⁹Pd, ¹¹¹Ag,¹¹¹In, ¹¹³In, ¹²¹Te, ¹²²Te, ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁵Te, ¹²⁶I, ¹³¹I, ¹³¹In,¹³³I, ¹⁴²Pr, ¹⁴³Pr, ¹⁵³Pb, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁵Tm, ¹⁶⁶Dy, ¹⁶⁶H, ¹⁶⁷Tm,¹⁶⁸Tm, ¹⁶⁹Yb, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁷Pt, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰¹Tl,²⁰³Hg, ²¹¹At, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁴Ac, and 225Ac.

II.D. Methods of Preparing PTK7 Antibody-Drug Conjugate

Also provided are methods for preparing antibody-drug conjugates of thepresent invention. For example, a process for producing a PTK7antibody-drug conjugate as disclosed herein can include (a) linking thelinker to the drug; (b) conjugating the linker-drug moiety to theantibody; and (c) purifying the antibody-drug conjugate. Representativemethods for synthesis of vc0101 and mc8261 are described in Example 9,and representative methods for conjugation of anti-PTK7-vc0101,anti-PTK7-mc8621 ADCs and anti-PTK7-AcButCM ADCs are described inExample 10.

In one aspect, an antibody-drug conjugate of the formula Ab-(L-D) may beprepared by (a) adding the linker-drug moiety (e.g. vc0101 or mc8261) toan anti-PTK7 antibody, or antigen-binding fragment thereof, whereinanti-PTK7 antibodies may be partially reduced in a solution containing:2-10 molar excess of tris(2-carboxyethyl)phosphine (TCEP), 100 mM of4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (HEPES) havinga pH of 6-9 and 1 mM diethylenetriaminepentaacetic acid (DTPA) for aperiod of time ranging from about 30 minutes to 16 hours at atemperature ranging from about 0-37 CC. The vc0101 or mc8261linker-payload may then be added at a linker-payload/antibody molarratio from about 4-10 with dimethylacetamide (DMA) for an incubationperiod of time ranging from about 30 minutes to 16 hours at atemperature ranging from about 0-37 CC. Subsequently, the unreactedthiols may be capped with N-ethylmaleimide and the unreactedlinker-payload may be quenched with L-Cys.

In another aspect, an antibody-drug conjugate of the formula Ab-(L-D maybe prepared by (a) adding the linker-drug moiety (e.g. AcButCM) to ananti-PTK7 antibody, or antigen-binding fragment thereof, wherein theconcentration of antibody may range from 1 to 25 mg/ml and thelinker-drug moiety is at a molar ratio ranging from about 1-15 to 1 ofthe anti-PTK7 antibody; (b) incubating the linker-drug moiety andanti-PTK7 antibody in a non-nucleophilic, protein-compatible, bufferedsolution having a pH in a range from about 7 to 9 to produce anmonomeric antibody-drug conjugate, wherein the solution furthercompromises (i) a suitable organic cosolvent, and (ii) an additivehaving at least one C₆-C₁₈ carboxylic acid or its salt, and wherein theincubation is conducted at a temperature ranging from about 00° C. toabout 45° C., for a period of time ranging from about 1 minute to about24 hours; and (c) subjecting the conjugate produced in step (b) to achromatographic separation process to separate antibody-drug conjugateswith a DAR from 1 to 8; and provides low conjugated fraction (LCF) ofbelow 10% from unconjugated anti-PTK7 antibody, linker-drug moiety, andaggregated conjugates.

Optimal reaction conditions for formation of a conjugate may beempirically determined by variation of reaction variables such astemperature, pH, linker-payload moiety input, and additiveconcentration. Conditions suitable for conjugation of other drugs may bedetermined by those skilled in the art without undue experimentation.

In some aspects the drug may be modified to include a group reactivewith a conjugation point on an antibody. For example, a drug may beattached by alkylation (e.g., at the epsilonamino group lysines or theN-terminus of antibodies), reductive amination of oxidized carbohydrate,transesterification between hydroxyl and carboxyl groups, amidation atamino groups or carboxyl groups, and conjugation to thiols. In someembodiments, the number of drug (D) molecules conjugated per antibodymolecule ranges from 1 to 8; 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or1 to 2. In other embodiments, the number of drug (D) moleculesconjugated per antibody is 1, 2, 3, 4, 5, 6, 7 or 8. In someembodiments, compositions, batches, and/or formulations of a pluralityof antibody-drug conjugates may be characterized by an average DAR. Theaverage DAR ranges from about 1 to about 8, about 1 to about 7, about 1to about 6, about 1 to about 5, about 1 to about 4; about 1 to about 3,about 1 to about 2. In some embodiments, the average DAR for acomposition, batch, and/or formulation of a plurality of antibody-drugconjugates ranges from about 2 to about 8, about 2 to about 7, about 2to about 6, about 2 to about 5, about 2 to about 4, about 2 to about 3or about 3 to about 5. As used in the foregoing ranges of average DAR,the term “about” means +/−0.5%. For examples of chemistries that can beused for conjugation, see, e.g., Current Protocols in Protein Science(John Wiley & Sons, Inc.), Chapter 15 (Chemical Modifications ofProteins).

Other methods for preparing antibody-drug conjugates have been describedin various publications. For example, chemical modification can be madein the antibodies either through lysine side chain amines or throughcysteine sulfhydryl groups activated by reducing interchain disulfidebonds for the conjugation reaction to occur. See, e.g., Tanaka et al.,FEBS Letters 579:2092-2096, 2005, and Gentle et al., Bioconjugate Chem.15:658-663, 2004. Further, reactive cysteine residues engineered atspecific sites of antibodies for specific drug conjugation with definedstoichiometry have also been described. See, e.g., Junutula et al.,Nature Biotechnology, 26:925-932, 2008.

Further as described in International Publication No. WO 2013/093809,certain residues presumably present on the surface of the CH2 or CH3domain of the heavy chain of antibodies, or on the constant domain ofthe light chain, or otherwise accessible, are suitable for thesubstitution of the naturally-occurring wild type amino acid with, forexample, cysteine, and are therefore useful to engineer a site capableof conjugation to various agents,

In some aspects, an engineered Fc polypeptide of the invention may beused to prepare a PTK7 antibody or antibody-drug conjugate, such thatthe antibody or fragment thereof thereby comprises an engineered Fcregion which can be used to conjugate, at the engineered residue (i.e.,the amino acid substituted compared to wild type unmodified Fc), a widevariety of agents.

The PTK7 antibodies and antibody-drug conjugates of the presentinvention may encompass an engineered Fc polypeptide where 1, 2, or moreamino acids chosen from positions: 347, 392, 398, 422 and 443 of theantibody heavy chain (HC) of a parent, native, or wild type antibody,substituted with another amino acid (including natural andnon-natural/synthetic amino acids), wherein the numbering system of theconstant region is that of the EU index according to Kabat.

It should be noted that a single substitution in an Fc polypeptide, forexample of a cysteine residue, normally results in the display of twocorresponding residues in the resultant IgG antibody due to thehomodimeric nature of IgG antibody molecules. Thus, the resultantengineered IgG antibodies of the invention may display at least 1, 2, 3,4, or more reactive groups for the purpose of conjugation to a drug orcompound. In an aspect, one or more of the substitutions is with acysteine residue, and the resulting engineered antibodies may display atleast 1, 2, 3, 4, or more thiol groups for the purpose of conjugation toa drug or compound.

In another aspect, an engineered Fc polypeptide of the disclosure maycomprise one or more substitutions selected from the positions 347, 392,398, 422 and 443, of the heavy chain (HC) of an antibody, wherein thenumbering system of the constant region is that of the EU index as setforth in Kabat, and wherein the amino acid sequence of the heavy chainis selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 37,and SEQ ID NO: 61.

The PTK7 antibodies and antibody-drug conjugates of the presentinvention may encompass an engineered antibody light chain constantregion (LC), or a portion thereof, where 1, 2, or 3 amino acids chosenfrom positions 111, 183, or 188, of the antibody light chain, whereinthe numbering system of the light chain constant region is that of theKabat, of a parent, native, or wild type antibody, substituted withanother amino acid (including natural and non-natural/synthetic aminoacids).

In some aspects, the engineered LC polypeptide of the disclosurecomprises one or more substitutions from positions 111, 183, or 188, ofthe antibody light chain wherein the amino acid sequence of the lightchain is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO:47, and SEQ ID NO: 71

In other aspects, due to the dimeric nature of many antibodies (e.g.,IgGs comprise two light chains and two heavy chains, each heavy chaincomprising an Fc polypeptide), an antibody of the invention may compriseat least one engineered Fc polypeptide and may further comprise at leastone engineered light chain constant polypeptide thereby providing atleast two site-specific conjugation sites—one in the Fc polypeptide andanother in the LC polypeptide.

In some aspects of the invention, the antibody, or antigen-bindingfragment thereof, of the disclosed PTK7 antibody-drug conjugatesincludes an IgG1 heavy chain constant region, for example a hu23 heavychain set forth as SEQ ID NO: 13, a hu24 heavy chain set forth as SEQ IDNO: 37, or a hu58 heavy chain set forth as SEQ ID NO: 61. In otheraspects, the antibody, or antigen-binding fragment thereof, of thedisclosed PTK7 antibody-drug conjugates includes a kappa light chainconstant region, for example a hu23 light chain set forth as SEQ ID NO:23, a hu24 light chain set forth as SEQ ID NO: 47, or a hu58 light chainset forth as SEQ ID NO: 71. In particular aspects of the invention, aPTK7 antibody-drug conjugate can include an IgG1 heavy chain constantregion and a kappa light chain constant region, for example, a heavychain set forth as SEQ ID NO: 13 and a light chain set forth as SEQ IDNO: 23; or as another example, a heavy chain set forth as SEQ ID NO: 37and a light chain set forth as SEQ ID NO: 47; or as another example, aheavy chain set forth as SEQ ID NO: 61 and a light chain set forth asSEQ ID NO: 71.

Also, as described in International Publication No. WO2012/059882,conjugation methods include use of an acyl donor glutamine-containingtag or an endogenous glutamine made reactive (i.e., the ability to forma covalent bond as an acyl donor) by polypeptide engineering in thepresence of transglutaminase and an amine (e.g., a cytotoxic agentcomprising or attached to a reactive amine).

In some aspects, the PTK7 antibody or antibody-drug conjugate maycomprise an acyl donor glutamine-containing tag engineered at a specificsite of the antibody (e.g., a carboxyl terminus, an amino terminus, orat another site) of the PTK7 antibody. In some aspects, the tagcomprises an amino acid glutamine (Q) or an amino acid sequence GGLLQGG(SEQ ID NO:74), LLQGA (SEQ ID NO:75), GGLLQGA (SEQ ID NO:76), LLQ,LLQGPGK (SEQ ID NO: 77), LLQGPG (SEQ ID NO: 78), LLQGPA (SEQ ID NO: 79),LLQGP (SEQ ID NO: 80), LLQP (SEQ ID NO: 81), LLQPGK (SEQ ID NO: 82),LLQGAPGK (SEQ ID NO: 83), LLQGAPG (SEQ ID NO: 84), LLQGAP (SEQ ID NO:85), LLQX₁X₂X₃X₄X₅, wherein X₁ is G or P, wherein X₂ is A, G, P, orabsent, wherein X₃ is A, G, K, P, or absent, wherein X₄ is G, K orabsent, and wherein X₅ is K or absent (SEQ ID NO: 86), or LLQX₁X₂X₃X₄X₅,wherein X₁ is any naturally occurring amino acid and wherein X₂, X₃, X₄,and X₅ are any naturally occurring amino acids or absent (SEQ ID NO:87). In some embodiments, the PTK7 antibody or antibody-drug conjugatemay comprise an amino acid substitution from asparagine (N) to glutamine(Q) at position 297 of the PTK7 antibody.

In another aspect, the PTK7 antibody or antibody-drug conjugate maycomprise an acyl donor glutamine-containing tag and an amino acidmodification at position 222, 340, or 370 of the antibody (EU numberingscheme), wherein the modification is an amino acid deletion, insertion,substitution, mutation, or any combination thereof. Accordingly, in someaspects, the PTK7 antibody or antibody-drug conjugate may comprise theacyl donor glutamine-containing tag (e.g., Q, GGLLQGG (SEQ ID NO:74),LLQGA (SEQ ID NO:75), GGLLQGA (SEQ ID NO:76), LLQ, LLQGPGK (SEQ ID NO:77), LLQGPG (SEQ ID NO: 78), LLQGPA (SEQ ID NO: 79), LLQGP (SEQ ID NO:80), LLQP (SEQ ID NO: 81), LLQPGK (SEQ ID NO: 82), LLQGAPGK (SEQ ID NO:83), LLQGAPG (SEQ ID NO: 84), LLQGAP (SEQ ID NO: 85), LLQX₁X₂X₃X₄X₅,wherein X₁ is G or P, wherein X₂ is A, G, P, or absent, wherein X₃ is A,G, K, P, or absent, wherein X₄ is G, K or absent, and wherein X₅ is K orabsent (SEQ ID NO: 86), or LLQX₁X₂X₃X₄X₅, wherein X₁ is any naturallyoccurring amino acid and wherein X₂, X₃, X₄, and X₅ are any naturallyoccurring amino acids or absent (SEQ ID NO: 87) conjugated at a specificsite (e.g., at a carboxyl terminus of the heavy or light chain or atanother site) of the PTK7 antibody and an amino acid modification atposition 222, 340, or 370 of the antibody (EU numbering scheme).

To further increase the number of drug molecules per antibody-drugconjugate, the drug may be conjugated to polyethylene glycol (PEG),including straight or branched polyethylene glycol polymers andmonomers. A PEG monomer is of the formula: —(CH₂CH₂O)—. Drugs and/orpeptide analogs may be bound to PEG directly or indirectly, i.e. throughappropriate spacer groups such as sugars. A PEG-antibody-drugcomposition may also include additional lipophilic and/or hydrophilicmoieties to facilitate drug stability and delivery to a target site invivo. Representative methods for preparing PEG-containing compositionsmay be found in U.S. Pat. Nos. 6,461,603; 6,309,633; and 5,648,095,among other places.

For example, to increase the amount of auristatin or calicheamicin inPTK7 antibody-drug conjugates disclosed herein, the antibody may beconjugated to PEG prior to conjugation with the drug, for example, usingPEG-SPA, PEG-SBA, or PEG-bis-maleimide. Antibody-drug conjugatesprepared using PEG may show reduced binding affinity for the targetantigen, but are still effective as a result of increased drug load.

Following conjugation, the conjugates may be separated and purified fromunconjugated reactants and/or aggregated forms of the conjugates byconventional methods. This can include processes such as size exclusionchromatography (SEC), ultrafiltration/diafiltration, ion exchangechromatography (IEC), chromatofocusing (CF) HPLC, FPLC, or SephacrylS-200 chromatography. The separation may also be accomplished byhydrophobic interaction chromatography (HIC). Suitable HIC mediaincludes Phenyl Sepharose 6 Fast Flow chromatographic medium, ButylSepharose 4 Fast Flow chromatographic medium, Octyl Sepharose 4 FastFlow chromatographic medium, Toyopearl Ether-650M chromatographicmedium, Macro-Prep methyl HIC medium or Macro-Prep t-Butyl HIC medium.

In some aspects of the invention, the separation may be performed usingButyl Sepharose 4 Fast Flow chromatographic medium. When using acustomized gradient, higher DAR species that remain bound to the columnare removed. In some aspects, the purification process may include acentrifuge cell removal step, optionally a Protein A affinity capturestep followed by one or two orthogonal chromatographic polishing steps,a virus filtration step, and a tangential flow filtration step forconcentration and formulation.

III. Functional Assays for Characterization of PTK7 Antibody-DrugConjugates

The present invention further discloses in vitro and in vivo assays tocharacterize activities of a PTK7 antibody-drug conjugate, includingPTK7 binding activity, cellular internalization following binding toPTK7 antigen presented on a cell surface, and targeting toPTK7-expressing cells in a subject. In some aspects of the invention,PTK7 antibody-drug conjugates are characterized by the neutralizing ordepleting aspects of the antibody, or antigen-binding fragment thereof.In some aspects of the invention, PTK7 antibody-drug conjugates arecharacterized by unexpected efficacy of a particular drug as compared tolack of efficacy of an alternate drug. In some aspects of the invention,PTK7 antibody-drug conjugates are characterized as outperforming astandard-of-care therapeutic agent having a same mode of action as thedrug.

Techniques for detecting binding of PTK7 antibody-drug conjugates to aPTK7 antigen, or other PTK7 antigen, are known in the art, including forexample, BIACORE® assays. Additional representative techniques includecentrifugation, affinity chromatography and other immunochemicalmethods. See e.g., Manson (1992) Immunochemical Protocols, Humana Press,Totowa, N.J., United States of America; Ishikawa (1999) Ultrasensitiveand Rapid Enzyme Immunoassay, Elsevier, Amsterdam/New York. Antigenbinding assays may be performed using isolated PTK7 antigen orPTK7-expressing cells.

The binding specificity of PTK7 antibody-drug conjugates may be furtherdescribed by definition of a binding epitope, i.e., identification ofresidues, including nonadjacent residues that participate in antigenbinding, and/or definition of residues that influence antigen binding.

Internalization of PTK7 antibody-drug conjugates by PTK7-expressingcells may be assayed by observing the amount of antibodies or conjugatesbound to the surface of the PTK7-expressing cells over time. See e.g.,Example 7. Selected PTK7 ligands or their isoforms may be present in asoluble form, and at least some PTK7 likely remains associated with thecell surface thereby allowing for internalization of the antibodiesdisclosed herein. Accordingly, anti-PTK7 antibody-drug conjugates of thepresent invention may be internalized by cells that express PTK7. Forexample, an anti-PTK7 antibody-drug conjugate that binds to PTK7 on thesurface of a tumor initiating cell may be internalized by the tumorinitiating cell. The number of ADC molecules internalized may besufficient or adequate to kill a PTK7 expressing cell, especially a PTK7expressing tumor cell. Depending on the potency of the ADC, in someinstances, the uptake of a single ADC molecule into the cell issufficient to kill the target cell to which the ADC binds. For example,certain toxins are highly potent in killing such that internalization ofone molecule of the toxin conjugated to the antibody is sufficient tokill the tumor cell.

Internalization of PTK7 antibodies may be assessed using a functionalassay in which cells are incubated with the PTK7 antibody and asecondary antibody Fab fragment that is conjugated to the saporin toxin.Cell viability is then measured by any suitable method, with cellularcytotoxicity indicative of antibody internalization. See Example 7.

In some aspects of the invention, the antibody, or antigen-bindingfragment thereof, of the disclosed PTK7 antibody-drug conjugates is an“antagonist” as used in the broadest sense, i.e., any molecule thatpartially or fully blocks, inhibits, or neutralizes a biologicalactivity of a native target disclosed herein or the transcription ortranslation thereof. The terms “inhibit” or “neutralize” as used hereinwith respect to bioactivity of an antibody of the invention mean theability of the antibody to substantially antagonize, prohibit, prevent,restrain, slow, disrupt, eliminate, stop, reduce or reverse e.g.progression or severity of that which is being inhibited including, butnot limited to, a biological activity. For example, in some aspects ofthe invention, anti-PTK7 antibody-drug conjugate facilitate cell killingupon internalization of the antibody-drug conjugate. For example, aneutralizing antibody or antagonist will preferably diminish a PTK7function by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%,95%, 97%, 99% or more.

In other aspects of the invention the anti-PTK7 antibody-drug conjugatesof the present invention may be depleting antibodies. The term depletingantibody refers to an antibody that binds to or associates with PTK7 onor near the cell surface and induces, promotes or causes the death orelimination of the cell (e.g., by complement-dependent cytotoxicity orantibody-dependent cellular cytotoxicity). Preferably a depletingantibody will be able to remove, eliminate or kill at least 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% of tumorperpetuating cells in a defined cell population.

Functional assays also include methods for assessing anti-canceractivity of antibody-drug conjugates, for example, an ability to destroyexisting cancer cells, or to delay or prevent growth of cancer cells.Cancers targeted by antibody-drug conjugates of the invention includeboth primary and metastasized tumors and carcinomas of any tissue in asubject, including carcinomas and hematopoietic malignancies such asleukemias and lymphomas.

PTK7 antibody-drug conjugates having growth inhibitory activity caneliminate PTK7-expressing cells or to prevent or reduce proliferation ofPTK7-expressing cancer cells. Representative methods for rapid in vitroassessment of cell growth inhibition are described in Jones et al.(2001) J. Immunol. Methods 254:85-98.

PTK7 antibody-drug conjugates may also include an ability to induce celldeath, for example, programmed cell death characterized by nuclear DNAdegradation, nuclear degeneration and condensation, loss of membraneintegrity, and phagocytosis. Representative assays to assess cell aredescribed in Hoves et al. (2003) Methods 31:127-34; Peng et al. (2002)Chin. Med. Sci. J. 17:17-21; Yasuhara et al. (2003) J. Histochem.Cytochem. 51:873-885.

For example, to assess the cytotoxicity of PTK7 antibody-drug conjugatesin vitro, PTK7-expressing cancer cells and control cells are cultured inthe presence PTK7 antibody-drug conjugates and separately with freedrug. The cytotoxicity of each agent is reported as ED50 (ng/ml), whichis the amount of drug given as conjugate or as free drug that causes 50%reduction of a cell culture relative to an untreated control. The numberof cells in culture is determined using a vital dye (MTS) following drugexposure. See Example 12.

To assess the cytotoxicity of PTK7 antibody-drug conjugates in vivo,tumors are prepared in NOD/SCID, nude (nu/nu) or other strain ofimmune-compromised mice by subcutaneous injection of various cancercells. PTK7 antibody-drug conjugates and control compounds may beadministered to tumor-bearing mice, for example, by intraperitonealinjection twice a week for two weeks (q4d×4). Measurable therapeuticoutcomes include inhibition of tumor cell growth. See Example 13.

Further, the present invention provides for PTK7 antibody-drugconjugates that may deplete, silence, neutralize, eliminate or inhibitgrowth, propagation or survival of tumor cells, including tumorinitiating cells (TIC), and/or associated neoplasia through a variety ofmechanisms, including agonizing or antagonizing selected pathways oreliminating specific cells depending, for example, on the anti-PTK7antibody, or dosing and method of delivery.

As used herein, the term tumor initiating cell (TIC) encompasses bothtumor perpetuating cells (TPC; i.e., cancer stem cells or CSC) andhighly proliferative tumor progenitor cells (TProg), which togethergenerally include a unique subpopulation (i.e. 0.1-40%) of a bulk tumoror mass. For the purposes of the instant disclosure the terms tumorperpetuating cells and cancer stem cells are equivalent and may be usedinterchangeably herein. Conversely, TPC differ from TProg in that theycan completely recapitulate the composition of tumor cells existingwithin a tumor and have unlimited self-renewal capacity as demonstratedby serial transplantation (two or more passages through mice) of lownumbers of isolated cells. As used herein, the term “tumor initiatingcell” also refers to cancer stem cells of various hematologicmalignancies, which are not characterized by a tumor per se.

The present invention provides PTK7 antibody-drug conjugates that targettumor initiating cells (TIC), and especially tumor perpetuating cells(TPC), thereby facilitating the treatment, management or prevention ofneoplastic disorders and hyperproliferative disorders. Morespecifically, specific tumor cell subpopulations express PTK7 and likelymodify localized coordination of morphogen signaling important to cancerstem cell self-renewal and cell survival. Thus, PTK7 antibody-drugconjugates may be used to reduce the frequency of TICs uponadministration to a subject. The reduction in tumor initiating cellfrequency may occur as a result of a) elimination, depletion,sensitization, silencing or inhibition of tumor initiating cells; b)controlling the growth, expansion or recurrence of tumor initiatingcells; c) interrupting the initiation, propagation, maintenance, orproliferation of tumor initiating cells; or d) by otherwise hinderingthe survival, regeneration and/or metastasis of the tumorigenic cells.In some aspects of the invention, the reduction in the frequency oftumor initiating cells occurs as a result of a change in one or morephysiological pathways. The change in the pathway, whether by reductionor elimination of the tumor initiating cells or by modifying theirpotential (e.g., induced differentiation, niche disruption) or otherwiseinterfering with their ability to exert effects on the tumor environmentor other cells, in turn allows for the more effective treatment ofPTK7-associated disorders by inhibiting tumorigenesis, tumor maintenanceand/or metastasis and recurrence.

Among the methods that can be used to assess such a reduction in thefrequency of tumor initiating cells is limiting dilution analysis eitherin vitro or in vivo, preferably followed by enumeration using Poissondistribution statistics or assessing the frequency of predefineddefinitive events such as the ability to generate tumors in vivo or not.It is also possible to determine reduction of frequency values throughwell-known flow cytometric or immunohistochemical means. As to all theaforementioned methods see, for example, Dylla et al. 2008, PMCID:PMC2413402 & Hoey et al. 2009, PMID: 19664991, each of which isincorporated herein by reference in its entirety. Other methodscompatible with the instant invention that may be used to calculatetumor initiating cell frequency, include quantifiable flow cytometrictechniques and immunohistochemical staining procedures.

Using any of the above-referenced methods it is then possible toquantify the reduction in frequency of TIC (or the TPC therein) providedby the disclosed PTK7 antibody-drug conjugates in accordance with theteachings herein. In some instances, the PTK7 antibody-drug conjugatesof the instant invention may reduce the frequency of TIC (by a varietyof mechanisms noted above, including elimination, induceddifferentiation, niche disruption, silencing, etc.) by 10%, 15%, 20%,25%, 30% or even by 35%. In other aspects of the invention, thereduction in frequency of TIC may be on the order of 40%, 45%, 50%, 55%,60% or 65%. In certain aspects of the invention, the disclosed compoundsmy reduce the frequency of TIC by 70%, 75%, 80%, 85%, 90% or even 95%.It will be appreciated that any reduction of the frequency of the TIClikely results in a corresponding reduction in the tumorigenicity,persistence, recurrence and aggressiveness of the neoplasia.

Amassing evidence supports the hypothesis that tumor growth, resistanceto therapy, and disorder relapse are controlled by TPC. The frequency ofTPC may vary in a tumor type or between patients with the same tumortype as a product of disorder stage and/or degree of differentiationwithin the tumor. TPC can be identified and enriched using panels ofcell surface markers that often overlap in their expression amongpatients with certain types of cancer. TPC are best defined by theirfunctional ability to initiate tumors upon serial transplantation,whereas non-tumorigenic (NTG) cells are devoid of this capacity. Solidtumor cells enriched for their unique tumor initiating capacity werefirst identified in breast cancer; however, breast cancer includes aspectrum of malignancies. To date, the scientific community has failedto associate specific TPC identities with particular disorder subtypes,which may underlie discrepant results both across and within groups andmay also increase the likelihood of failed translation to the clinic.

The present invention provides a combination of new cell surface makersthat improve the enrichment of TPC. In a particular aspect, theinvention provides a combination of new cell surface makers thatfacilitate the enrichment of triple negative breast cancer (TNBC) TPC.The present invention further provides for the identification of PTK7 asa novel TPC-associated therapeutic target in TNBC; the expression levelof which is significantly higher than in other breast cancer subtypesand normal tissue.

The pharmacokinetics of PTK7 antibody-drug conjugates can be evaluatedand compared to the pharmacokinetics of unconjugated antibody in variousanimals. For example, this can be done following a single intravenousbolus administration in female NOD/SCID, nude (nu/nu) or other strain ofimmune-compromised mice, male Sprague-Dawley rats, and female cynomolgusmonkeys. Pharmacokinetics of PTK7 antibody-drug conjugates are generallycharacterized by low clearance, low volume of distribution, and longapparent terminal half-life in various species. The serum concentrationsof unconjugated auristatin derivatives are expected to be below thequantification limit. The toxicity profile for these conjugates insingle-dose toxicity ranging studies is expected to be similar to thatobtained for other antibody-drug conjugates at comparable doses.

An antibody, antibody-drug conjugate or other agent which “inducesapoptosis” is one which induces programmed cell death as determined bybinding of annexin V, fragmentation of DNA, cell shrinkage, dilation ofendoplasmic reticulum, cell fragmentation, and/or formation of membranevesicles (called apoptotic bodies). The cell is a tumor cell, e.g.,breast, ovarian, colorectal, prostate, liver and lung. Various methodsare available for evaluating the cellular events associated withapoptosis. For example, phosphatidyl serine (PS) translocation can bemeasured by annexin binding; DNA fragmentation can be evaluated throughDNA laddering; and nuclear/chromatin condensation along with DNAfragmentation can be evaluated by any increase in hypodiploid cells.

As used herein “antibody-dependent cell-mediated cytotoxicity” or “ADCC”refers to a cell-mediated reaction in which nonspecific cytotoxic cellsthat express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in an animalmodel such as that disclosed in Clynes et al., PNAS (USA), 95:652-656(1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202:163-171 (1997), maybe performed.

“Human effector cells” are leukocytes that express one or more FcRs andperform effector functions. The cells may express FcγRIII and carry outantigen-dependent cell-mediated cyotoxicity (ADCC) effector function.Examples of human leukocytes that mediate ADCC include but are notlimited to peripheral blood mononuclear cells (PBMC), natural killer(NK) cells, monocytes, macrophages, eosinophils, and neutrophils, withPBMCs and NK cells being preferred. Antibodies that have ADCC activityare typically of the IgG1 or IgG3 isotype. Such ADCC-mediatingantibodies can also be made by engineering a variable region from anon-ADCC antibody or variable region fragment to an IgG1 or IgG3 isotypeconstant region.

IV. Uses of PTK7 Antibody-Drug Conjugates

The antibodies and the antibody drug-conjugates of the present inventionare useful in various applications including, but are not limited to,therapeutic treatment methods and diagnostic treatment methods.

IV.A. In Vitro Applications

The present invention provides in vitro methods using PTK7 antibody-drugconjugates. For example, the disclosed antibodies may be used, eitheralone or in combination with cytotoxic agents or other drugs tospecifically bind PTK7-positive cancer cells to deplete such cells froma cell sample. Methods are also provided for inducing apoptosis and/orinhibition of cell proliferation via contacting PTK7-expressing cellswith a PTK7 antibody-drug conjugate. Representative in vitro methods aredescribed herein above under the heading of “Functional Assays forCharacterization of PTK7 antibody-drug conjugates.”

PTK7 antibody-drug conjugates of the invention also have utility in thedetection of PTK7-positive cells in vitro based on their ability tospecifically bind PTK7 antigen. A method for detecting PTK7-expressingcells may include: (a) preparing a biological sample having cells; (b)contacting a PTK7 antibody-drug conjugates with the biological sample invitro, wherein the drug is a detectable label; and (c) detecting bindingthe PTK7 antibody-drug conjugates.

PTK7 antibody-drug conjugates disclosed herein are also useful forreducing the frequency of tumor initiating cells in a tumor sample. Forexample, the method can include the steps contacting in vitro a tumorcell population, wherein the population comprises tumor initiating cellsand tumor cells other than tumor initiating cells, with a PTK7antibody-drug conjugate; whereby the percentage of tumor initiatingcells in the cell population is reduced. As used herein, the term “tumorinitiating cell” also refers to cancer stem cells of various hematologicmalignancies, which are not characterized by a tumor per se.Representative tumor samples include any biological or clinical samplewhich contains tumor cells, for example, a tissue sample, a biopsy, ablood sample, plasma, saliva, urine, seminal fluid, etc.

IV.B. Therapeutic Applications

PTK7 associated disorders or conditions include but are not limited tomesothelioma, hepatobiliary (hepatic and biliary duct), hepatocellularcarcinoma, a primary or secondary CNS tumor, a primary or secondarybrain tumor, lung cancer (NSCLC and SCLC), bone cancer, pancreaticcancer, skin cancer, cancer of the head or neck, melanoma, ovariancancer, colon cancer, rectal cancer, cancer of the anal region, stomachcancer, gastrointestinal (such as gastric, colorectal, and duodenalcancers), breast cancer (such as triple-negative breast cancer (TNBC),progesterone-receptor positive breast cancer (PR+), estrogen-receptorpositive breast cancer (ER+) and double positive breast cancer), uterinecancer, carcinoma of the fallopian tubes, carcinoma of the endometrium,carcinoma of the cervix, carcinoma of the vagina, carcinoma of thevulva, Hodgkin's Disease, esophageal cancer, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, prostatecancer, testicular cancer, leukemias (such as acute myeloid leukemia(AML) and acute lymphoblastic leukemia (ALL), chronic myeloid leukemia),lymphocytic lymphomas, cancer of the bladder, cancer of the kidney orureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasmsof the central nervous system (CNS), primary CNS lymphoma, non-Hodgkin'slymphoma, spinal axis tumors, brain stem glioma, pituitary adenoma,adrenocortical cancer, gall bladder cancer, multiple myeloma,cholangiocarcinoma, fibrosarcoma, neuroblastoma, retinoblastoma, and allcancers represented by the PTK7-expressing cell types shown in Tables 8and 9 or a combination of one or more of the cancers disclosed herein.

The phrase “effective amount”, “effective dosage” or as used hereinrefers to an amount of a drug, compound or pharmaceutical compositionnecessary to achieve any one or more beneficial or desired therapeuticresults. For prophylactic use, beneficial or desired results includeeliminating or reducing the risk, lessening the severity, or delayingthe outset of the disorder, including biochemical, histological and/orbehavioral symptoms of the disorder, its complications and intermediatepathological phenotypes presenting during development of the disorder.For therapeutic use, beneficial or desired results include clinicalresults such as reducing incidence or amelioration of one or moresymptoms of various PTK7 associated disorders decreasing the dose ofother medications required to treat the disorder, enhancing the effectof another medication, and/or delaying the progression of the PTK7associated disorder of patients.

In one aspect, the invention provides a method for treating a disorderassociated with PTK7 expression in a subject. The invention alsoprovides an antibody-drug conjugate, or a pharmaceutical composition, asdescribed herein, for use in a method for treating a disorder associatedwith PTK7 expression in a subject. The invention further provides theuse of an antibody-drug conjugate, or a pharmaceutical composition, asdescribed herein, in the manufacture of a medicament for treating adisorder associated with PTK7 expression in a subject.

In some aspects of the invention, the method of treating a disorderassociated with PTK7 expression in a subject includes administering tothe subject in need thereof an effective amount of a composition (e.g.,pharmaceutical composition) having the PTK7 antibody-drug conjugates asdescribed herein. The disorders associated with PTK7 expression include,but are not limited to, abnormal PTK7 expression, altered or aberrantPTK7 expression, PTK7 overexpression, and a proliferative disorder(e.g., cancer).

In one aspect of the invention, the disorder is cancer, including, butnot limited to, mesothelioma, hepatobiliary (hepatic and biliary duct),hepatocellular carcinoma, a primary or secondary CNS tumor, a primary orsecondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, melanoma,ovarian cancer, colon cancer, rectal cancer, cancer of the anal region,stomach cancer, gastrointestinal (such as gastric, colorectal, andduodenal cancers), breast cancer (such as triple-negative breast cancer(TNBC), progesterone-receptor positive breast cancer (PR+),estrogen-receptor positive breast cancer (ER+) and double positivebreast cancer), uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, Hodgkin's Disease, esophageal cancer,cancer of the small intestine, cancer of the endocrine system, cancer ofthe thyroid gland, cancer of the parathyroid gland, cancer of theadrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer ofthe penis, prostate cancer, testicular cancer, leukemias (such as acutemyeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), chronicmyeloid leukemia), lymphocytic lymphomas, cancer of the bladder, cancerof the kidney or ureter, renal cell carcinoma, carcinoma of the renalpelvis, neoplasms of the central nervous system (CNS), primary CNSlymphoma, non-Hodgkin's lymphoma, spinal axis tumors, brain stem glioma,pituitary adenoma, adrenocortical cancer, gall bladder cancer, multiplemyeloma, cholangiocarcinoma, fibrosarcoma, neuroblastoma,retinoblastoma, and all cancers represented by the PTK7-expressing celltypes shown in Tables 8 and 9 or a combination of one or more of thecancers disclosed herein.

In another embodiment, cancers suitable for targeting using theanti-PTK7 antibody-drug conjugate include PTK7-expressing primary andmetastatic cancers, such as breast cancer (such as triple-negativebreast cancer (TNBC), progesterone-receptor positive breast cancer(PR+), estrogen-receptor positive breast cancer (ER+) and doublepositive breast cancer); ovarian cancer; colorectal cancer; esophagealcancer; gastric cancer; melanoma; sarcoma; kidney cancer; pancreaticcancer; prostate cancer; liver cancer, such as hepatocellular carcinoma(HCC); and lung cancer, such as non-small cell lung cancer (NSCLC) andsmall cell lung cancer (SCLC).

In a more specific embodiment, cancers suitable for targeting usinganti-PTK7 antibody-drug conjugates include PTK7-expressing primary andmetastatic cancers, such as breast cancer (such as triple-negativebreast cancer (TNBC), progesterone-receptor positive breast cancer(PR+), estrogen-receptor positive breast cancer (ER+) and doublepositive breast cancer) NSCLC, prostate cancer and esophageal cancer. Ina more specific embodiment, cancers suitable for targeting usinganti-PTK7 antibody-drug conjugates include PTK7-expressing primary andmetastatic cancers, such as breast cancer (such as triple-negativebreast cancer (TNBC)) and NSCLC.

In some aspects of the invention, provided is a method of inhibitingtumor growth or progression in a subject who has a PTK7 expressingtumor, including administering to the subject in need thereof aneffective amount of a composition having the PTK7 antibody-drugconjugates as described herein. In other aspects of the invention,provided is a method of inhibiting metastasis of PTK7 expressing cancercells in a subject, including administering to the subject in needthereof an effective amount of a composition having the PTK7antibody-drug conjugates as described herein. In other aspects of theinvention, provided is a method of inducing regression of a PTK7expressing tumor regression in a subject, including administering to thesubject in need thereof an effective amount of a composition having thePTK7 antibody-drug conjugates as described herein. In other aspects, theinvention provides an antibody-drug conjugate, or a pharmaceuticalcomposition, as described herein, for use in a method as describedabove. In other aspects the invention provides the use of anantibody-drug conjugate, or a pharmaceutical composition, as describedherein, in the manufacture of a medicament for use in the methodsdescribed above.

Thus, patients to be treated with PTK7 antibody-drug conjugates of theinvention may be selected based on biomarker expression, including butnot limited to mRNA (qPCR) of bulk tumor samples and elevated expressionof PTK7 antigen which results in a patient population selected forenriched target expression rather than tumor origin or histology. Targetexpression can be measured as a function of the number of cells stainingcombined with the intensity of the cells staining. For example,classification of high expression of PTK7 includes those patients withgreater than 30% (i.e., 40%, 50% or 60%) of the cells tested byimmunohistochemical staining positive for PTK7 at a level of 3+(on ascale of 1 to 4), while moderate expression of the PTK7 can includethose patients with greater than 20% of the cell cells staining at 1+ to2+. Target expression can also be measured by detecting PTK7 expressionon tumor initiating cells (TIC) as described herein.

Biomarkers other than expression of PTK7 can be also used for patientselection, including characterization of the tumor based on multi-drugresistance (MDR), for example. Nearly 50% of human cancers are eithercompletely resistant to chemotherapy or respond only transiently, afterwhich they are no longer affected by commonly used anticancer drugs.This phenomenon is referred to as MDR and is inherently expressed bysome tumor types, while others acquire MDR after exposure tochemotherapy treatment. The drug efflux pump β-glycoprotein mediates amajority of the MDR associated with cytotoxic chemotherapeutics.Phenotypic and functional analysis of MDR mechanisms present in cancerpatient tumor specimens can be conducted in order to relate specific MDRmechanism(s) with resistance to chemotherapy in specific tumor types.

Cancer growth or abnormal proliferation refers to any one of a number ofindices that suggest change within cells to a more developed cancer formor disease state. Inhibition of growth of cancer cells or cells of anon-neoplastic proliferative disorder may be assayed by methods known inthe art, such as delayed tumor growth and inhibition of metastasis.Other indices for measuring inhibition of cancer growth include adecrease in cancer cell survival, a decrease in tumor volume ormorphology (for example, as determined using computed tomographic (CT),sonography, or other imaging method), destruction of tumor vasculature,improved performance in delayed hypersensitivity skin test, an increasein the activity of cytolytic T-lymphocytes, and a decrease in levels oftumor-specific antigens.

Desired outcomes of the disclosed therapeutic methods are generallyquantifiable measures as compared to a control or baseline measurement.As used herein, relative terms such as “improve,” “increase,” or“reduce” indicate values relative to a control, such as a measurement inthe same individual prior to initiation of treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. Arepresentative control individual is an individual afflicted with thesame form of hyperproliferative disorder as the individual beingtreated, who is about the same age as the individual being treated (toensure that the stages of the disorder in the treated individual and thecontrol individual are comparable.

Changes or improvements in response to therapy are generallystatistically significant. As used herein, the term “significance” or“significant” relates to a statistical analysis of the probability thatthere is a non-random association between two or more entities. Todetermine whether or not a relationship is “significant” or has“significance,” statistical manipulations of the data can be “p-value.”Those p-values that fall below a user-defined cut-off point are regardedas significant. A p-value less than or equal to 0.1, less than 0.05,less than 0.01, less than 0.005, or less than 0.001 may be regarded assignificant.

As described herein above under the heading “111. Functional Assays forCharacterization of PTK7 Antibody-Drug Conjugates,” the presentinvention also provides methods for targeting tumor initiating cells.More particularly, PTK7 antibody-drug conjugates of the invention maydeplete, silence, neutralize, eliminate or inhibit growth, propagationor survival of tumor cells, including tumor initiating cells.

Thus, PTK7 antibody-drug conjugates disclosed herein are also useful forreducing the frequency of tumor initiating cells in a tumor sample. Forexample, the method can include the steps contacting a tumor cellpopulation, wherein the population comprises tumor initiating cells andtumor cells other than tumor initiating cells, with a PTK7 antibody-drugconjugate; whereby the percentage of tumor initiating cells in the cellpopulation is reduced. As used herein, the term “tumor initiating cell”also refers to cancer stem cells of various hematologic malignancies,which are not characterized by a tumor per se. The contacting step maybe performed in vitro, wherein the tumor cell population is contained ina biological sample, as described herein above. Alternatively, thecontacting step may be performed in vivo as occurs followingadministration of a PTK7 antibody-drug conjugate to a subject.

IV.C. In Vivo Detection and Diagnosis

In another aspect, provided is a method of detecting, diagnosing, and/ormonitoring a disorder associated with PTK7 expression. For example, thePTK7 antibodies as described herein can be labeled with a detectablemoiety such as an imaging agent and an enzyme-substrate label. Theantibodies as described herein can also be used for in vivo diagnosticassays, such as in vivo imaging (e.g., PET or SPECT), or a stainingreagent.

Following administration of a PTK7 antibody-drug conjugate to a subject,wherein the drug is a detectable label, and after a time sufficient forbinding, the biodistribution of PTK7-expressing cells bound by theantibody may be visualized. The disclosed diagnostic methods may be usedin combination with treatment methods. In addition, PTK7 antibody-drugconjugates of the invention may be administered for the dual purpose ofdetection and therapy.

Representative non-invasive detection methods include scintigraphy(e.g., SPECT (Single Photon Emission Computed Tomography), PET (PositronEmission Tomography), gamma camera imaging, and rectilinear scanning),magnetic resonance imaging (e.g., convention magnetic resonance imaging,magnetization transfer imaging (MTI), proton magnetic resonancespectroscopy (MRS), diffusion-weighted imaging (DWI) and functional MRimaging (fMRI)), and ultrasound.

IV.D. Formulation

The present invention further provides pharmaceutical compositionsincluding any of the PTK7 antibody-drug conjugates disclosed herein anda pharmaceutically acceptable carrier. Further, the compositions caninclude more than one PTK7 antibody or PTK7 antibody-drug conjugate(e.g., a mixture of PTK7 antibodies that recognize different epitopes ofPTK7). Other exemplary compositions include more than one PTK7 antibodyor PTK7 antibody-drug conjugate that recognize the same epitope(s), ordifferent species of PTK7 antibodies or PTK7 antibody-drug conjugatethat bind to different epitopes of PTK7 (e.g., human PTK7).

The composition used in the present invention can further includepharmaceutically acceptable carriers, excipients, or stabilizers(Remington: The Science and practice of Pharmacy 21st Ed., 2005,Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations, and may include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrans; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). “Pharmaceutically acceptable salt” as usedherein refers to pharmaceutically acceptable organic or inorganic saltsof a molecule or macromolecule. Pharmaceutically acceptable excipientsare further described herein.

Various formulations of the PTK7 antibody or the PTK7 antibody-drugconjugate may be used for administration. In some aspects of theinvention, the PTK7 antibody or the PTK7 antibody-drug conjugate may beadministered neat. The PTK7 antibody or the PTK7 antibody-drug conjugateand a pharmaceutically acceptable excipient may be in variousformulations. Pharmaceutically acceptable excipients are known in theart, and are relatively inert substances that facilitate administrationof a pharmacologically effective substance. For example, an excipientcan give form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. Excipients as well asformulations for parenteral and nonparenteral drug delivery are setforth in Remington, The Science and Practice of Pharmacy 20th Ed. MackPublishing, 2000.

In some aspects of the invention, these agents are formulated foradministration by injection (e.g., intraperitoneally, intravenously,subcutaneously, intramuscularly, etc.). Accordingly, these agents can becombined with pharmaceutically acceptable vehicles such as saline,Ringer's solution, dextrose solution, and the like. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history.

Therapeutic formulations of the PTK7 antibody or the PTK7 antibody-drugconjugate used in accordance with the present invention are prepared forstorage by mixing an antibody having the desired degree of purity withoptional pharmaceutically acceptable carriers, excipients or stabilizers(Remington, The Science and Practice of Pharmacy 21st Ed. MackPublishing, 2005), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and mayinclude buffers such as phosphate, citrate, and other organic acids;salts such as sodium chloride; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens, such asmethyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Liposomes containing the PTK7 antibody or the PTK7 antibody-drugconjugate are prepared by methods known in the art, such as described inEppstein, et al., Proc. Natl. Acad. Sci. USA 82:3688-3692 (1985); Hwang,et al., Proc. Natl Acad. Sci. USA 77:4030-4034 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition including phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington, The Science and Practice of Pharmacy 21st Ed. MackPublishing, 2005.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or ‘poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by, for example, filtration through sterilefiltration membranes. Therapeutic PTK7 antibody or PTK7 antibody-drugconjugate compositions are generally placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle.

The compositions according to the present invention may be in unitdosage forms such as tablets, pills, capsules, powders, granules,solutions or suspensions, or suppositories, for oral, parenteral orrectal administration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical carrier, e.g. conventionaltableting ingredients such as corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother pharmaceutical diluents, e.g. water, to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill caninclude an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g. TWEEN™ 20, 40, 60, 80 or 85) andother sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently include between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as INTRALIPID™, LIPOSYN™, INFONUTROL™, LIPOFUNDIN™ andLIPIPHYSAN™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g. egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%. The fat emulsion can include fat dropletsbetween 0.1 and 1.0 μm, particularly 0.1 and 0.5 μm, and have a pH inthe range of 5.5 to 8.0.

The emulsion compositions can be those prepared by mixing a PTK7antibody or a PTK7 antibody-drug conjugate with INTRALIPID™ or thecomponents thereof (soybean oil, egg phospholipids, glycerol and water).

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as set outabove. In some aspects of the invention, the compositions areadministered by the oral or nasal respiratory route for local orsystemic effect. Compositions in preferably sterile pharmaceuticallyacceptable solvents may be nebulised by use of gases. Nebulisedsolutions may be breathed directly from the nebulising device or thenebulising device may be attached to a face mask, tent or intermittentpositive pressure breathing machine. Solution, suspension or powdercompositions may be administered, preferably orally or nasally, fromdevices which deliver the formulation in an appropriate manner.

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers including the PTK7 antibodyor the PTK7 antibody-drug conjugate as described herein and instructionsfor use in accordance with any of the methods of the invention describedherein. Generally, these instructions include a description ofadministration of the PTK7 antibody or the PTK7 antibody-drug conjugatefor the above described therapeutic treatments.

The instructions relating to the use of the PTK7 antibodies or the PTK7antibody conjugates as described herein generally include information asto dosage, dosing schedule, and route of administration for the intendedtreatment. The containers may be unit doses, bulk packages (e.g.,multi-dose packages) or sub-unit doses. Instructions supplied in thekits of the invention are typically written instructions on a label orpackage insert (e.g., a paper sheet included in the kit), butmachine-readable instructions (e.g., instructions carried on a magneticor optical storage disk) are also acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a PTK7 antibody or PTK7 antibody-drug conjugate. Thecontainer may further include a second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit includes a container and alabel or package insert(s) on or associated with the container.

IV.E. Dose and Administration

For in vitro and in vivo applications, PTK7 antibody-drug conjugates areprovided or administered in an effective dosage. In a clinical context,an effective dosage of drug, compound, or pharmaceutical composition isan amount sufficient to accomplish prophylactic or therapeutic treatmenteither directly or indirectly. An effective dosage can be administeredin one or more administrations. An effective dosage of a drug, compound,or pharmaceutical composition may or may not be achieved in conjunctionwith another drug, compound, or pharmaceutical composition. Thus, an“effective dosage” may be considered in the context of administering oneor more therapeutic agents, and a single agent may be considered to begiven in an effective amount if, in conjunction with one or more otheragents, a desirable result may be or is achieved. For detection ofPTK7-positive cells using the disclosed PTK7 antibody-drug conjugates, adetectable amount of a composition of the invention is administered to asubject, i.e., a dose of the conjugate such that the presence of theconjugate may be determined in vitro or in vivo.

For example, when administered to a cancer-bearing subject, an effectiveamount includes an amount sufficient to elicit anti-cancer activity,including cancer cell cytolysis, inhibition of cancer cellproliferation, induction of cancer cell apoptosis, reduction of cancercell antigens, delayed tumor growth, and/or inhibition of metastasis.Tumor shrinkage is well accepted as a clinical surrogate marker forefficacy. Another well accepted marker for efficacy is progression-freesurvival.

The PTK7 antibody or the PTK7 antibody-drug conjugates can beadministered to an individual via any suitable route. It should beunderstood by persons skilled in the art that the examples describedherein are not intended to be limiting but to be illustrative of thetechniques available. Accordingly, in some aspects of the invention, thePTK7 antibody or the PTK7 antibody conjugate is administered to anindividual in accord with known methods, such as intravenousadministration, e.g., as a bolus or by continuous infusion over a periodof time, by intramuscular, intraperitoneal, intracerebrospinal,intracranial, transdermal, subcutaneous, intra-articular, sublingually,intrasynovial, via insufflation, intrathecal, oral, inhalation ortopical routes. Administration can be systemic, e.g., intravenousadministration, or localized. Commercially available nebulizers forliquid formulations, including jet nebulizers and ultrasonic nebulizersare useful for administration. Liquid formulations can be directlynebulized and lyophilized powder can be nebulized after reconstitution.Alternatively, the PTK7 antibody or the PTK7 antibody-drug conjugate canbe aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

In some aspects of the invention, the PTK7 antibody or the PTK7antibody-drug conjugate is administered via site-specific or targetedlocal delivery techniques. Examples of site-specific or targeted localdelivery techniques include various implantable depot sources of thePTK7 antibody or the PTK7 antibody-drug conjugate or local deliverycatheters, such as infusion catheters, indwelling catheters, or needlecatheters, synthetic grafts, adventitial wraps, shunts and stents orother implantable devices, site specific carriers, direct injection, ordirect application. See, e.g. PCT International Publication No. WO2000/53211 and U.S. Pat. No. 5,981,568.

PTK7 antibodies or the PTK7 antibody-drug conjugates as described hereincan be administered using any suitable method, including by injection(e.g., intraperitoneally, intravenously, subcutaneously,intramuscularly, etc.). The PTK7 antibody or the PTK7 antibody-drugconjugate can also be administered via inhalation, as described herein.Generally, for administration of a PTK7 antibody and a PTK7antibody-drug conjugate, an initial candidate dosage can be about 2mg/kg. For the purpose of the present invention, a typical daily dosagemight range from about any of 3 μg/kg to 30 μg/kg to 300 μg/kg to 3mg/kg, to 30 mg/kg, to 100 mg/kg or more, depending on the factorsmentioned above. For example, dosage of about 1 mg/kg, about 2.5 mg/kg,about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. Forrepeated administrations over several days or longer, depending on thedisorder, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved, forexample, to inhibit or delay tumor growth/progression or metastases ofcancer cells. An exemplary dosing regimen includes administering aninitial dose of about 2 mg/kg, followed by a weekly maintenance dose ofabout 1 mg/kg of the PTK7 antibody or PTK7 antibody-drug conjugate, orfollowed by a maintenance dose of about 1 mg/kg every other week. Otherexemplary dosing regimens include administering increasing doses (e.g.,initial dose of 1 mg/kg and gradual increase to one or more higher dosesevery week or longer time period). Other dosage regimens may also beuseful, depending on the pattern of pharmacokinetic decay that thepractitioner wishes to achieve. For example, in some aspects of theinvention, dosing from one to four times a week is contemplated. Inother aspects, dosing once a month or once every other month or everythree months is contemplated, as well as weekly, bi-weekly and everythree weeks. The progress of this therapy may be easily monitored byconventional techniques and assays. The dosing regimen (including thePTK7 antibody or the PTK7 antibody-drug conjugate used) can vary overtime.

For the purpose of the present invention, the appropriate dosage of aPTK7 antibody or a PTK7 antibody-drug conjugate will depend on the PTK7antibody or the PTK7 antibody-drug conjugate (or compositions thereof)employed, the type and severity of symptoms to be treated, whether theagent is administered for therapeutic purposes, previous therapy, thepatient's clinical history and response to the agent, the patient'sclearance rate for the administered agent, and the discretion of theattending physician. The clinician may administer a PTK7 antibody or aPTK7 antibody-drug conjugate until a dosage is reached that achieves thedesired result and beyond. Dose and/or frequency can vary over course oftreatment, but may stay constant as well. Empirical considerations, suchas the half-life, generally will contribute to the determination of thedosage. For example, antibodies that are compatible with the humanimmune system, such as humanized antibodies or fully human antibodies,may be used to prolong half-life of the antibody and to prevent theantibody being attacked by the host's immune system. Frequency ofadministration may be determined and adjusted over the course oftherapy, and is generally, but not necessarily, based on treatmentand/or suppression and/or amelioration and/or delay of symptoms, e.g.,tumor growth inhibition or delay, etc. Alternatively, sustainedcontinuous release formulations of PTK7 antibodies or PTK7 antibody-drugconjugates may be appropriate. Various formulations and devices forachieving sustained release are known in the art.

In some aspects of the invention, dosages for a PTK7 antibody or a PTK7antibody-drug conjugate may be determined empirically in individuals whohave been given one or more administration(s) of the PTK7 antibody orthe PTK7 antibody-drug conjugate. Individuals are given incrementaldosages of a PTK7 antibody or a PTK7 antibody-drug conjugate. To assessefficacy, an indicator of the disorder can be followed.

Administration of a PTK7 antibody or a PTK7 antibody-drug conjugate inaccordance with the method in the present invention can be continuous orintermittent, depending, for example, upon the recipient's physiologicaldisorder, whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of a PTK7 antibody or a PTK7 antibody-drug conjugate maybe essentially continuous over a preselected period of time or may be ina series of spaced doses.

IV.F. Combination Therapies

In some aspects of the invention, the methods described herein furtherinclude a step of treating a subject with an additional form of therapy.In some aspects, the additional form of therapy is an additionalanti-cancer therapy including, but not limited to, chemotherapy,radiation, surgery, hormone therapy, and/or additional immunotherapy.

The disclosed PTK7 antibody-drug conjugates may be administered as aninitial treatment, or for treatment of disorders that are unresponsiveto conventional therapies. In addition, the PTK7 antibody-drugconjugates may be used in combination with other therapies (e.g.,surgical excision, radiation, additional anti-cancer drugs etc.) tothereby elicit additive or potentiated therapeutic effects and/or reducehepatocytotoxicity of some anti-cancer agents. PTK7 antibody-drugconjugates of the invention may be co-administered or co-formulated withadditional agents, or formulated for consecutive administration withadditional agents in any order.

Representative agents useful for combination therapy include any of thedrugs described herein above as useful for preparation of PTK7antibody-drug conjugates under the subheading “Drugs.” PTK7antibody-drug conjugates of the invention may also be used incombination with other therapeutic antibodies and antibody-drugconjugates, including anti-PTK7 antibodies other than the disclosedanti-PTK7 antibodies, as well as antibodies and conjugates targeting adifferent antigen. Representative antibodies, which may be used alone oras an antibody-drug conjugate, include anti-5T4 antibodies (e.g., A1,A2, and A3), anti-CD19 antibodies, anti-CD20 antibodies (e.g., RITUXAN®,ZEVALIN®, BEXXAR®), anti-CD22 antibodies, anti-CD33 antibodies (e.g.,MYLOTARG®), anti-CD33 antibody-drug conjugates, anti-Lewis Y antibodies(e.g., Hu3S193, Mthu3S193, AGmthu3S193), anti-HER-2 antibodies (e.g.,HERCEPTIN® (trastuzumab), MDX-210, OMNITARG® (pertuzumab, rhuMAb 2C4)),anti-CD52 antibodies (e.g., CAMPATH®), anti-EGFR antibodies (e.g.,ERBITUX® (cetuximab), ABX-EGF (panitumumab)), anti-VEGF antibodies(e.g., AVASTIN® (bevacizumab)), anti-DNA/histone complex antibodies(e.g., ch-TNT-1/b), anti-CEA antibodies (e.g., CEA-Cide, YMB-1003)hLM609, anti-CD47 antibodies (e.g., 6H9), anti-VEGFR2 (or kinase insertdomain-containing receptor, KDR) antibodies (e.g., IMC-1C11),anti-Ep-CAM antibodies (e.g., ING-1), anti-FAP antibodies (e.g.,sibrotuzumab), anti-DR4 antibodies (e.g., TRAIL-R), anti-progesteronereceptor antibodies (e.g., 2C5), anti-CA19.9 antibodies (e.g., GIVAREX®)and anti-fibrin antibodies (e.g., MH-1).

The disclosed PTK7 antibody-drug conjugates may also be administeredtogether with one or more combinations of cytotoxic agents as part of atreatment regimen. Useful cytotoxic preparations for this purposeinclude CHOPP (cyclophosphamide, doxorubicin, vincristine, prednisoneand procarbazine); CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone); COP (cyclophosphamide, vincristine, prednisone); CAP-BOP(cyclophosphamide, doxorubicin, procarbazine, bleomycin, vincristine andprednisone); m-BACOD (methotrexate, bleomycin, doxorubicin,cyclophosphamide, vincristine, dexamethasone, and leucovorin;ProMACE-MOPP (prednisone, methotrexate, doxorubicin, cyclophosphamide,etoposide, leukovorin, mechloethamine, vincristine, prednisone andprocarbazine); ProMACE-CytaBOM (prednisone, methotrexate, doxorubicin,cyclophosphamide, etoposide, leukovorin, cytarabine, bleomycin andvincristine); MACOP-B (methotrexate, doxorubicin, cyclophosphamide,vincristine, prednisone, bleomycin and leukovorin); MOPP(mechloethamine, vincristine, prednisone and procarbazine); ABVD(adriamycin/doxorubicin, bleomycin, vinblastine and dacarbazine); MOPP(mechloethamine, vincristine, prednisone and procarbazine) alternatingwith ABV (adriamycin/doxorubicin, bleomycin, vinblastine); MOPP(mechloethamine, vincristine, prednisone and procarbazin) alternatingwith ABVD (adriamycin/doxorubicin, bleomycin, vinblastine anddacarbazine); ChlVPP (chlorambucil, vinblastine, procarbazine,prednisone); IMVP-16 (ifosfamide, methotrexate, etoposide); MIME(methyl-gag, ifosfamide, methotrexate, etoposide); DHAP (dexamethasone,high-dose cytaribine and cisplatin); ESHAP (etoposide,methylpredisolone, HD cytarabine, and cisplatin); CEPP(B)(cyclophosphamide, etoposide, procarbazine, prednisone and bleomycin);CAMP (lomustine, mitoxantrone, cytarabine and prednisone); and CVP-1(cyclophosphamide, vincristine and prednisone); DHAP (cisplatin,high-dose cytarabine and dexamethasone); CAP (cyclophosphamide,doxorubicin, cisplatin); PV (cisplatin, vinblastine or vindesine); CE(carboplatin, etoposide); EP (etoposide, cisplatin); MVP (mitomycin,vinblastine or vindesine, cisplatin); PFL (cisplatin, 5-fluorouracil,leucovorin); IM (ifosfamide, mitomycin); IE (ifosfamide, etoposide); IP(ifosfamide, cisplatin); MIP (mitomycin, ifosfamide, cisplatin); ICE(ifosfamide, carboplatin, etoposide); PIE (cisplatin, ifosfamide,etoposide); Viorelbine and cisplatin; Carboplatin and paclitaxel; CAV(cyclophosphamide, doxorubicin, vincristine); CAE (cyclophosphamide,doxorubicin, etoposide); CAVE (cyclophosphamide, doxorubicin,vincristine, etoposide); EP (etoposide, cisplatin); and CMCcV(cyclophosphamide, methotrexate, lomustine, vincristine).

PTK7 antibody-drug conjugates may be used in combination with systemicanti-cancer drugs, such as epithilones (BMS-247550, Epo-906),reformulations of taxanes (Abraxane, Xyotax), microtubulin inhibitors(MST-997, TTI-237), or with targeted cytotoxins such as CMD-193 andSGN-15. Additional useful anti-cancer agents include TAXOTERE®,TARCEVA®, GEMZAR® (gemcitabine), 5-FU, AVASTIN® ERBITUX®, TROVAX®,anatumomab mafenatox, letrazole, docetaxel, and anthracyclines.

For combination therapies, a PTK7 antibody-drug conjugate and/or one ormore additional therapeutic or diagnostic agents are administered withinany time frame suitable for performance of the intended therapy ordiagnosis. Thus, the single agents may be administered substantiallysimultaneously (i.e., as a single formulation or within minutes orhours) or consecutively in any order. For example, single agenttreatments may be administered within about 1 year of each other, suchas within about 10, 8, 6, 4, or 2 months, or within 4, 3, 2 or 1week(s), or within about 5, 4, 3, 2 or 1 day(s). The administration of aPTK7 antibody-drug conjugate in combination with a second therapeuticagent preferably elicits a greater effect than administration of eitheralone.

In some aspects of the invention, the additional form of therapyincludes administering one or more therapeutic agent in addition to thePTK7 antibodies or the PTK7 antibody-drug conjugates as describedherein. The therapeutic agents include, but are not limited to, a secondantibody (e.g., an anti-VEGF antibody, an anti-HER2 antibody, anti-CD25antibody, and/or an anti-CD20 antibody), an angiogenesis inhibitor, acytotoxic agent, an anti-inflammatory agent (e.g., paclitaxel,docetaxel, cisplatin, doxorubicin, prednisone, mitomycin, progesterone,tamoxifen, or fluorouracil).

In some aspects of the invention, more than one PTK7 antibody or PTK7antibody-drug conjugate may be present. At least one, at least two, atleast three, at least four, at least five different or more PTK7antibody or PTK7 antibody-drug conjugate can be present. Generally,those PTK7 antibodies or PTK7 antibody-drug conjugates may havecomplementary activities that do not adversely affect each other. Forexample, one or more of the following PTK7 antibody may be used: a firstPTK7 antibody directed to one epitope on PTK7 and a second PTK7 antibodydirected to a different epitope on PTK7.

The disclosed combination therapies may elicit a synergistic therapeuticeffect, i.e., an effect greater than the sum of their individual effectsor therapeutic outcomes. Measurable therapeutic outcomes are describedherein. For example, a synergistic therapeutic effect may be an effectof at least about two-fold greater than the therapeutic effect elicitedby a single agent, or the sum of the therapeutic effects elicited by thesingle agents of a given combination, or at least about five-foldgreater, or at least about ten-fold greater, or at least abouttwenty-fold greater, or at least about fifty-fold greater, or at leastabout one hundred-fold greater. A synergistic therapeutic effect mayalso be observed as an increase in therapeutic effect of at least 10%compared to the therapeutic effect elicited by a single agent, or thesum of the therapeutic effects elicited by the single agents of a givencombination, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or more. A synergistic effect is also aneffect that permits reduced dosing of therapeutic agents when they areused in combination.

As used throughout the detailed description, the term “about” means avalue+/−1% of the value following the term “about,” unless otherwiseindicated.

EXAMPLES

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

Example 1 Generation and Humanization of Anti-PTK7 Antibodies

PTK-7 antibodies in the form of murine antibodies were produced inaccordance with procedures known in the art and as described in PCTInternational Publication No. WO 2012/112943. Murine antibodiesgenerated were humanized using complementarity determining region (CDR)grafting. Human frameworks for heavy and light chains were selectedbased on sequence and structure similarity with respect to functionalhuman germline genes. Structural similarity was evaluated by comparingthe mouse canonical CDR structure to human candidates with the samecanonical structures as described in Chothia et al. (supra).

More particularly, murine antibodies designated herein mu23, mu24, andmu58 described in PCT International Publication No. WO 2012/112943 werehumanized using a computer aided CDR-grafting method (Abysis Database,UCL Business Plc.) and standard molecular engineering techniques toprovide humanized mu23, mu24, and mu58, hereinafter hu23, hu24, andhu58, respectively. The human framework regions of the variable regionswere selected based on their highest sequence homology to the mouseframework sequence and its canonical structure. For the purposes of theanalysis, the assignment of amino acids to each of the CDR domains wasin accordance with the Kabat et al. numbering. Several humanizedantibody variants were made in order to generate the optimal humanizedantibody with the humanized antibodies generally retaining theantigen-binding complementarity determining regions (CDRs) from themouse hybridoma in association with human framework regions. Hu23, hu24,and hu58 mAbs bound to the human PTK7 antigen with similar affinity totheir murine counterparts as measured using the BIACORE® system.

Molecular engineering procedures were conducted using art recognizedtechniques. Total mRNA was extracted from the hybridomas according tothe manufacturer's protocol (TRIZOL® Plus RNA Purification System, LifeTechnologies). A sequence specific 5′ leader sequence primer, designedto amplify each hybridoma, was used in combination with 3′ human Cylprimer to amplify and clone the variable regions of each humanizedantibody. Similarly a 5′ Vk leader sequence designed specifically toamplify each of the Vk regions combined with a single reverse primerspecific to the human kappa constant region were used to amplify andclone the kappa light chain. The amplified fragments were cloned aschimeric human gamma1/kappa chains and served as a bench mark for eachhumanized mAb.

From the nucleotide sequence information, data regarding V, D and J genesegments of the heavy and light chains of murine antibodies mu23, mu24,and mu58 were obtained. Based on the sequence data, new primer setsspecific to the leader sequence of the Ig VH and Vk chain of theantibodies were designed for cloning of the recombinant monoclonalantibody. Subsequently the V-(D)-J sequences were aligned with mouse Iggerm line sequences.

Heavy chain genes of mu23 were identified as VH3609 (V), DSP2.3 (D) andJH3. The heavy chain genes of mu24 were identified as VHJ558 (V), DSP2.7(D) and JH4. The heavy chain genes of mu58 were identified as IGHV 4-1(V), DFL 16.1 (D) and JH4. All three light chains were K class. Lightchain genes were identified as IGKVI4-111 and JK5 for the mu23, IGKV3-5and JK1 for the mu24, and IGKV17-121 and JK4 germ line sequences formu58. These results are summarized in the Table 2 below.

TABLE 2 Clone VH DH JH VL JL mu23 VH3609 DSP2.3 JH3 IGKVI4-111 JK5 mu24VHJ558 DSP2.7 JH4 IGKV3-5 JKI mu58 IGHV4-1 DFL16.1 JH4 IGKV17-121 JK4The obtained heavy and light chain sequences from all three clones werealigned to the functional human variable region sequences and reviewedfor homology and canonical structure. The results of the humanized heavyand light chain analysis are shown below in Tables 3 and 4 respectively,for the humanized anti-PTK7 antibodies hu23, hu24, and hu58.

TABLE 3 % % homology homology to human to mouse Humanized human humanhuman germ line germ line mAb VH DH JH sequence sequence hu23 VH2-5IGHD5-5 JH4 91 81 hu24 VH1-3 IGHD4-23 JH6 82 82 hu58 VH3-7 IGHD2-8 JH686 88

TABLE 4 % homology to human % homology Humanized human human germ lineto mouse mAb VK JK sequence sequence hu23 O8 JK5 91 81 hu24 L6 JK1 82 82hu58 B2 JK4 86 88

The amino acid sequences and associated nucleic acid sequence of hu23,hu24 and hu58 are shown above in Table 1 above. The amino acid sequencesof the VH region for hu23, hu24, and hu58 are shown in SEQ ID NO: 1, SEQID NO: 25, and SEQ ID NO: 49 respectively, with the correspondingnucleic acid sequences set forth in SEQ ID NO: 2, SEQ ID NO: 26, and SEQID NO: 50 respectively. The amino acid sequence of the kappa VL regionof hu23, hu24, and hu58 are shown in SEQ ID NO: 15, SEQ ID NO: 39, andSEQ ID NO: 63 respectively, with the corresponding nucleic acidsequences set forth in SEQ ID NO: 16, SEQ ID NO: 40, and SEQ ID NO: 64respectively.

As demonstrated in the Examples below each of the aforementionedhumanized antibodies functions as an effective anti-PTK7 antibody-drugconjugate in accordance with the teachings herein.

Example 2 Expression of Humanized Antibodies

The anti-PTK7 antibodies hu23, hu24, and hu58 were expressed andisolated using art recognized techniques and as described in PCTInternational Publication No. WO 2012/112943. Synthetic humanizedvariable DNA fragments (Integrated DNA Technologies) of the heavy chainswere cloned into human IgG 1 expression vectors. The variable lightchain fragments were cloned into human C-kappa expression vectors. Eachantibody was expressed by co-transfection of the corresponding heavy andthe light chain into CHO cells.

More particularly, for antibody production, directional cloning of thehumanized variable gene PCR products into human immunoglobulinexpression vectors was undertaken. All primers used in Ig gene-specificPCRs included restriction sites, AgeI and XhoI for IgH, XmaI and DraIIIfor Igk, which allowed direct cloning into expression vectors containingthe human IgG 1, and Igk constant regions, respectively. In brief, PCRproducts were purified with Qiaquick PCR purification kit (Qiagen, Inc.)followed by digestion with AgeI and XhoI (IgH), XmaI and DraIII (Igk),respectively. Digested PCR products were purified prior to ligation intoexpression vectors. Ligation reactions were performed in a total volumeof 10 μL with 200U T4-DNA Ligase (New England Biolabs), 7.5 μL ofdigested and purified gene-specific PCR product and 25 ng linearizedvector DNA. Competent E. coli DH10B bacteria (Life Technologies) weretransformed via heat shock at 42° C. with 3 μL ligation product andplated onto ampicillin plates (100 μg/mL). The AgeI-EcoRI fragment ofthe VH region was than inserted into the same sites of pEE6.4HuIgG1(Lonza AG) expression vector while the synthetic XmaI-DraIII VK insertwas cloned into the XmaI-DraIII sites of the respective pEE12.4Hu-Kappaexpression vector.

Cells producing humanized antibodies were generated by transfection ofHEK 293 cells with the appropriate plasmids using 293fectin. Plasmid DNAwas purified with QIAprep Spin columns (Qiagen). Human embryonic kidney(HEK) 293T (ATCC No CRL-11268) cells were cultured in 150 mm plates(Falcon, Becton Dickinson) under standard conditions in Dulbecco'sModified Eagle's Medium (DMEM) supplemented with 10% heat inactivatedFCS, 100 μglmL streptomycin, 100 U/mL penicillin G (all from LifeTechnologies).

For transient transfections cells were grown to 80% confluency. Equalamounts of IgH and corresponding IgL chain vector DNA (12.5 μg of eachvector DNA) was added to 1.5 mL OptiMEM mixed with 50 μL HEK 293transfection reagent in 1.5 mL opti-MEM. The mix was incubated for 30minutes at room temperature and distributed evenly to the culture plate.Supernatants were harvested three days after transfection, replaced by20 mL of fresh DMEM supplemented with 10% FBS and harvested again at day6 after transfection. Culture supernatants were cleared from cell debrisby centrifugation at 800×g for 10 minutes and stored at 4° C.Recombinant chimeric and humanized antibodies were purified with ProteinG beads (GE Healthcare). Further purification of hu23 and hu24 by ionexchange chromatography was required in order to achieve consistent,reproducible bioconjugation to vc0101 and mc8261. Without the additionalpurification step, the efficiency of thiol reduction and thus theresulting ADC drug-to-antibody ratio (DAR), described in Example 10,fluctuated dramatically and unpredictably. The requirement for theadditional purification was not anticipated but was determinedempirically.

Example 3 Characterization of Hu24 Binding

A comparison of the chimeric and humanized clone 24 mAbs was determinedby SPR using a Biacore™ 2000 (GE Healthcare). An antihuman antibodycapture kit was used to immobilize capture mAbs on a CM5 biosensor chip.Prior to each antigen injection cycle, humanized mAb at a concentrationof 2 g/mL was captured on the surface with a contact time of 2 minutesand a flow rate of 5 L/minute. The captured mAb loading from baselinewas constant at 80-120 response units. Following test article captureand 1 minute baseline, monomeric human PTK7 protein was flowed over thesurface at concentrations of 50, 25, and 12.5 nM for a 2-minuteassociation phase followed by a 2-minute dissociation phase at a flowrate of 5 L/minute. Following each cycle, the anti-human capture surfacewas regenerated with 30 seconds contact time of 3M MgCl₂ at 10 L/minute.

Biacore™ data was processed by initially subtracting a control IgGsurface from the specific mAb binding surface. The response data wasthen truncated to the association and dissociation phase. The resultingresponse curves with three different antigen concentrations were used tofit a 1:1 Langmuir binding model and to generate an apparent affinity bythe calculated K_(on) and K_(off) kinetics constants, with equilibriumdissociation constant defined as Kd=K_(off)/K_(on). All data analysissteps were completed in BiaEvaluation Software 3.1 (GE Healthcare).

The calculated affinity and kinetic constants were determined to bewithin 2-fold between the chimeric and humanized mAbs (Table 5).

TABLE 5 Affinity Constants of Chimeric and Humanized clone 24 mAbs toHuman PTK7 Test mAb K_(on) (M⁻¹s⁻¹) K_(off) (s⁻¹) Kd (nM) Chimeric clone24 3.9E+05 2.3E−04 0.6 Humanized clone 2.7E+05 3.1E−04 1.2 24 (hu24)K_(on) = Association rate constant; K_(off) = Dissociation rateconstant; Kd = Equilibrium dissociation constant; M = Molar; mAb =Monoclonal antibody; s = Seconds; nM = Nanomolar; Recombinant human PTK7ectodomain was used.

In order to determine the epitope recognized by hu24 mAb, its binding toseveral variants of the PTK7 ectodomain was evaluated. The PTK7ectodomain is comprised of 7 Ig domains, and the variants were designedto include two or more contiguous Ig domains. Variants were expressed asFc fusion proteins, and hu24 binding was determined by ELISA.

Constructs were designed with primers that amplified various contiguousPTK7 Ig domains as predicted by structural homology. The resultingsequences were fused in-frame with and upstream of the humanimmunoglobulin G2 (IgG2) Fc domain using standard molecular biologytechniques. The Fc fusion proteins were transfected into mammalian cellsand supernatants were harvested 72 hours later. Anti-PTK7 mAb hu24 wastested by ELISA for its ability to bind to the PTK7 protein variantswith defined Ig domains.

The results show that Ig domains 1-4 are required for hu24 binding toPTK7 (Table 6) and imply that the mAb recognizes tertiary structuralcharacteristics of PTK7.

TABLE 6 Binding of hu24 mAb to PTK7 Domains PTK7 ECD Ig Domains inConstruct 1-2 2-3 1-4 1-5 3-7 6-7 1-7 hu24 mAb binding − − + + − − + ECD= Extracellular Domains. Ig = Immunoglobulin. Domains 1-2 = Residue31-236; Domains 2-3 = Residues 110-321; Domains 1-4 = Residues 31-409;Domains 1-5 = Residues 31-510; Domains 3-7 = Residues 230-703; Domains6-7 = Residues 503-703; Domains 1-7 = Full length ECD, Residues 31-703.

Experiments were performed to characterize the cell binding propertiesof anti-PTK7 mAb hu24. To confirm antigen specificity, binding wasevaluated in cell lines with either substantial or negligible PTK7expression as determined by immunoblotting.

Whole cell extracts were resolved by gel electrophoresis and transferredto nitrocellulose membrane. The membrane was incubated with hu6M024 inTris-buffered saline with 0.1% Tween-20 (TBST)) with 5% weight/volumenon-fat milk, then washed with TBST, incubated with horseradishperoxidase-conjugated goat anti-human antibody (Santa Cruz BiotechnologyNo sc-2453) and washed extensively before exposure to a chemiluminscentsubstrate.

Immunoblotting indicated substantial expression of PTK7 in cell linesBxPC3 and MDAMB436 and negligible expression of PTK7 in cell line ASPC1(FIG. 2A). The flow cytometry based cell binding results with hu24 werefully consistent with the immunoblotting data (FIG. 2B) whichdemonstrated the antigen specificity of hu24.

Additional flow cytometry experiments were conducted to evaluate bindingof hu24 mAb to cancer cell lines with endogenous expression of PTK7.Briefly, adherent cells were dissociated with TrypLE™ Express (Gibco® No12604-021) which was then neutralized with culture media. Suspensioncells were harvested by centrifugation. Cells were resuspended instaining buffer (PBS with 3% BSA) with the stated concentration of mAband incubated on ice for 30 minutes. Cells were washed in stainingbuffer, resuspended in staining buffer with phycoerythrin (PE)-labeledanti-human antibody (Jackson ImmunoResearch No 109-115-098) andincubated on ice for 30 minutes. Cells were washed, resuspended instaining buffer with 7-AAD viability stain (BD Biosciences, PharmingenNo 51-68981 E) and analyzed by flow cytometry with a BD FACSCalibur™.The mean fluorescence intensity (MFI) in the PE channel of the viablecell population was determined for each sample.

Hu24 mAb exhibited binding to various cancer cell lines at the lowestconcentration tested (0.1 μg/ml). In contrast, the control antibody didnot show appreciable binding at the highest concentration tested (10g/ml) (Table 7).

TABLE 7 Binding Properties of hu24 mAb to Cancer Cell Lines MFI, ControlCancer Cell Line mAb MFI, hu6M024 mAb (tumor type) 10 μg/mL 0.1 μg/mL 1μg/mL 10 μg/mL H661 (lung cancer) 4.2 60 243 309 H446 (lung cancer) 1195 342 411 U2OS (osteosarcoma) 5.3 122 385 431 mAb = monoclonalantibody; MFI = mean fluorescence intensity; μg/mL = micrograms permilliliter.

Example 4 Expression of PTK7 in Various Cancer Cell Lines

Anti-PTK7 antibodies hu23 and hu24 exhibited specific binding tocultured cancer cell lines that were established from a broad range oftumor types, including solid and hematological indications, see Table 8below. Adherent cells were dissociated using TrypLE Express (GIBCO),neutralized with cell culture media and counted. Cells were plated intoa U-bottom 96-well plate with 5×10⁵ cells/100 μL media/well. The platewas centrifuged at 300×g, for 5 minutes at 4° C. to pellet cells and thesupernatant was discarded. Each pellet was resuspended in 10 μg/mL hu23,hu24 or non-binding control antibody in 3% BSA in PBS, and the plate wasincubated on ice for 30 minutes. The plate was centrifuged and the cellpellets were washed in 200 μL ice-cold 3% BSA in PBS. Each cell pelletwas resuspended in 100 μL of R-phycoerythrin (PE)-conjugated goatanti-human IgG Fc fragment that had been diluted 1:50 in 3% BSA in PBS,and the plate was incubated on ice for 30 minutes. The plate wascentrifuged and the cell pellets were washed in 200 μL of 3% BSA in PBSat 4° C. Each pellet was resuspended in 100 μL 3% BSA in PBS andtransferred to a 5 mL polycarbonate tube containing 250 μL 3% BSA inPBS. The samples were analyzed by flow cytometry using 5 μL7-Amino-Actinomycin D (7-AAD) staining solution per sample as aviability stain. Non-viable cells were excluded from the analysis.

The data in Table 8 shows mean fluorescent intensities (MFI) of antibodybinding to cancer cell lines by flow cytometry. Cell binding withhumanized antibodies hu23 and hu24 indicates PTK7 expression in numerouscell lines. PTK7 expression is prominent in various non-small cell lungcancer (NSCLC), small cell lung cancer (SCLC), colon, breast,pancreatic, and erythroleukemic cancer cell lines. A negative controlantibody that does not bind to PTK7 was used for comparison.

TABLE 8 Mean Fluorescent Intensity (10 μg/ml Ab) Negative Cell LineTumor type of origin hu24 hu23 Control Ab H520 Lung (NSCLC) 1384 12124.8 H446 Lung (SCLC) 937 915 7.1 H1048 Lung (SCLC) 955 900 4.4 DMS114Lung (SCLC) 793 568 9.9 HCT116 Colon 629 436 17.8 H69 Lung (SCLC) 404391 11.2 MDAMB468 Breast (BR) 364 302 12 MDAMB361-DYT2 Breast (BR) 295227 8.7 BxPC3 Pancreatic 207 192 4.6 MDAMB436 Breast (BR) 189 171 3.5H1299 Lung (NSCLC) 70.9 59.2 3 SKBR3 Breast (BR) 39.7 31.2 14.9 DU4475Breast (BR) 697 ND 3.8 DMS79 Lung (SCLC) 682 ND 8.5 H522 Lung (NSCLC)672 ND 3.8 MiaPaca Pancreatic 611 ND 3.5 H358 Lung (NSCLC) 474 ND 4.5HCC70 Breast (BR) 474 ND 9.4 H1975 Lung (NSCLC) 444 ND 4.8 H526 Lung(NSCLC) 438 ND 12.2 H661 Lung (NSCLC) 376 ND 3.8 HCC1937 Breast (BR) 370ND 7.8 H596 Lung (NSCLC) 355 ND 21.4 HCC827 Lung (NSCLC) 251 ND 5.8Hs700T Pancreatic 241 ND 4.9 HCC38 Breast (BR) 227 ND 7.2 TF1aErythroleukemia (AML) 208 ND 6.2 H2110 Lung (NSCLC) 171 ND 4.9 KG1Erythroleukemia (AML) 159 ND 5.2 TF1 Erythroleukemia (AML) 135 ND 5.9Hs578T Breast (BR) 124 ND 2.7 BT-549 Breast (BR) 114 ND 3.6 HCC1806Breast (BR) 77.5 ND 4.6 Kasumi-1 Erythroleukemia (AML) 68.2 ND 5.3 K562Chronic Myelogenous leukemia (CML) 56.1 ND 54.7 HEL Erythroleukemia(AML) 46 ND 15.7 RL Non-Hodgkin's lymphoma (NHL) 44.2 ND 29.8 RajiNon-Hodgkin's lymphoma (NHL) 36.3 ND 20.3 HL60 MX2 Promyelocyticleukemia (AML) 25.7 ND 25.2 HEL92.1 Erythroleukemia (AML) 23 ND 7.7 NB4Promyelocytic leukemia (AML) 19.5 ND 6.8 RPMI8226 Multiple myeloma 17.8ND 8.3 HL60 Promyelocytic leukemia (AML) 12.7 ND 10.9 MV411 Monocyticleukemia 10.1 ND 7.7 U937 Monocytic leukemia 5.8 ND 5.0 ASPC1 Pancreatic4.5 ND 2.6 THP-1 Monocytic leukemia 4.0 ND 4.0 EKVX Lung (NSCLC) 3.8 ND3.1 ND = no data.

Microarray data shown in Table 9 for 29 PDX tumor lines was generatedusing Agilent SurePrint GE 8×60 v2 arrays using total RNA isolated fromPDX tumor cells. Processing of the raw microarray data collected with asingle color included background subtraction and quantile normalization.Normalized data was log base 2 transformed, generating gene expressionvalues for use in downstream analyses. This data reflects the relativeamount of PTK7 expression associated with the indicated PDX cell line.BR=Breast, LU-Lung, OV=Ovarian, SK=Melanoma, CR=Colorectal, LIV=Liver.

TABLE 9 Relative PDX Cell Line mRNA level BR13 484.4 BR22 714.1 BR31210.8 BR56 393.4 BR64 237.2 BR120 319.6 BR36 324.0 BR133 639.1 LU176643.6 LU135 576.0 LU58 113.8 OV39 238.9 OV45 410.1 OV55 364.6 SK23 464.7SK25 171.3 SK19 484.4 LU86 288.0 LU95 377.4 LU64 247.3 LU49 56.5 LU70221.3 LU50 195.4 CR2 286.0 CR14 187.4 CR42 471.1 CR88 143.0 LIV13 75.6LIV40 254.2

PTK7 expression was also evaluated by immunohistochemistry in seven PDXmodels in immune compromised mice: four triple-negative breast cancerPDXs (BR13, BR22, BR31 and BR5); one progesterone receptor positive(PR+) breast cancer PDX (BR36); and two NSCLC PDXs (NSCLC135 andNSCLC176).

Briefly, a tissue fragment from each xenograft was formalin-fixed,processed and paraffin embedded (FFPE) using standard histologicalprocedures. Five-micron sections were cut onto charged slides, dried,deparaffinized in xylene and rehydrated with graded alcohols todistilled water. Heat-induced epitope retrieval was performed in BorgDecloaker (Biocare Medical) using a Retriever 2100 pressure cooker(Electron Microscopy Sciences) and cooled to room temperature (RT) for20 minutes (min). Endogenous peroxidase was quenched with Peroxidazed 1(Biocare Medical) for 10 min at RT. Non-specific protein interactionswere blocked with Background Punisher (Biocare Medical) for 10 min atRT. Tissue sections were incubated with primary antibody at 0.5 g/mL for1 hour at RT. Primary antibodies were either rabbit anti-PTK7 clone(Stem CentRx™ Inc) or rabbit isotype control (DA1E) mAb immunoglobulin G(IgG) XP® (Cell Signaling Technologies no. 3900). Binding of primaryantibody was detected with SignalStain® Boost IHC Detection Reagent(Cell Signaling Technologies no. 8114) for 30 min at RT. Staining wasdeveloped with DAB+ (3′,3′-Diaminobenzidine; DAKO) for 5 min at RT.Slides were briefly counterstained in CAT hematoxylin (Biocare Medical),washed in water, dehydrated in graded alcohols, cleared in xylene, andcoverslipped with Permount™ Mounting Medium (Fisher Chemicals).

PTK7 was observed on the plasma membrane in all of the PDX models (FIG.3).

Example 5 Expression of PTK7 in Various Tumor Tissues

PTK7 mRNA expression was determined in primary human tumors. Briefly,frozen tumor and normal tissues were fragmented, and mRNA was isolatedwith Qiagen RNeasy Mini kit (Qiagen, cat#74106). RNA quantitation andquality assessment was performed using the HT RNA microfluidic LabChipassay and LabChip GX microfluidic capillary electrophoresis instrument(Perkin Elmer). RNA for each of the samples was diluted so that thequantity fell within the linear range of the instrument (25-250 ng/μL).Isolated RNA samples were reverse transcribed to cDNA using the LifeTechnologies, High Capacity RNA-to-cDNA Kit (cat #4387406) following aprotocol outlined in the manufacturer's directions. The qRT-PCR reactionwas performed using the TaqMan Probe-Based Gene Expression Analysis andABI ViiA7 Real-Time PCR Systems (Life Technologies). Target gene andendogenous controls were run in quadruplicate for each probe set onpre-fabricated TaqMan low density array cards. ExpressionSuite Softwarev1.0.3 (Life Technologies) was used to generate automated thresholdvalues for signal amplification for a majority of samples. Rarely wereautomated thresholds adjusted manually. Amplification plots resulting inCt values >35 were discarded, as were those plots that generated a Ctvalue but did not display a trend of logarithmic amplification. All Ctvalues were exported from the ExpressionSuite software and relativequantification calculations were performed in Microsoft Excel 2010(Microsoft Corporation, Inc).

FIGS. 4A-C show the levels of PTK7 mRNA in (A) breast, (B) NSLC and (C)ovarian cancers. Quantitation of PTK7 expression was assessed using therelative fold difference (RQ) or comparative Ct method, (2^(−ΔΔct))method using the equation RQ=2^(−ΔΔct) The RQ data represents folddifference PTK7 expression relative to control RNA samples. For breastcarcinoma samples a normal breast RNA sample purchased from BioChain(Newark, Calif., cat# R1234086-50, lot# B610189, 75 year old female) wasused to generate RQ data. For lung cancer the RQ data reportedrepresents fold differences in PTK7 expression relative to normal lungRNA purchased from Life Technologies (cat #AM7968, lot #1308017, 80 yearold female). For ovarian carcinoma samples the RQ data reportedrepresents fold differences in PTK7 expression relative to RNA isolatedfrom normal ovary tissue (tissue ID #0204C011C) provided by theCleveland Clinic (Cleveland, Ohio). RQ values for all tumors was alsocalculated relative to a RNA pool from normal human tissues (BioChain,cat#R4234565, lot #B611043), as well as to a Universal Human ReferenceRNA pool (Agilent, cat #740000), which is comprised of equal parts RNAfrom 10 unique cancer cell lines.

Breast, NSCLC and ovarian tumors showed increased PTK7 mRNA expressionas compared to the corresponding normal tissue (FIGS. 4A-C. Theoverexpression in the TNBC was most notable, while overexpression inovarian tumors was modest.

FIG. 5 shows a correlation between higher PTK7 mRNA expression and worseoverall survival in NSCLC patients. To determine whether PTK7 expressionis associated with survival endpoint in lung cancer, Kaplan-Meieranalysis was applied to bioinformatics dataset with freewarehttp://kmplot.com/analysis (Gyorffy et al., 2013, PloS One. 18;8(12):e82241). PTK7 mRNA levels and patient survival data were plottedfor 719 NSCLC-adenocarcinoma patients using the tool's auto-select bestcutoff. High PTK7 expression was associated with shorter survival(hazard ratio HR=4.06, logrank P=1.1E-16).

PTK7 protein expression was seen in esophageal cancer. A tissuemicroarray (ES1502 from US Biomax) was used for immunohistochemistry.Briefly, sections from formalin-fixed paraffin-embedded tumor blockswere cut at 5 microns and baked onto glass slides. The slides werecleared in Xylene and rehydrated in graded alcohol washes ending inde-ionized water. The slides were retrieved in pH6 Citrate HIER bufferin the Retriever 2100 (Electron Microscopy). Peroxidazed, a hydrogenperoxide block solution (Biocare Medical), was applied to the slides for10 min. The slides were washed with TBST 2×, followed by BackgroundPunisher, a protein block, (Biocare Medical) for 10 min. The primaryantibody, H.235 (Lot #: 110325MM, Stock concentration: 12.7 mg/mL) wasapplied for 60 min at a concentration of 2 ug/mL. After washing withTBST (2×), the secondary antibody, DAKO anti-mouse Envision+, wasapplied for 30 min. After washing again with TBST (2×), the slides weredeveloped with Betazoid DAB+(Biocare Medical) for 5 min. The slides werethen counterstained in CAT Hematoxylin (Biocare Medical) for 30 secondsand coverslipped.

Forty out of 70 tumor samples scored positive for PTK7 expression on thecell membrane. Of the 40 samples that were PTK7 positive, 1 exhibitedhigh expression, 11 exhibited moderate expression and 28 exhibited lowexpression.

PTK7 protein expression was seen in prostate cancer. A tissue microarray(BC19013 from Biomax) was used for immunohistochemistry as describedabove for esophageal cancer. Eleven out of 26 tumor samples scoredpositive for PTK7 expression. Of the 11 samples that were PTK7 positive,2 exhibited moderate expression and 9 exhibited low expression.

Example 6 Measurement of PTK7 Protein in Serum

Reports in the literature have characterized cleavage of PTK7 at theplasma membrane which results in the shedding of part of theextracellular domain (Golubkov et al., 2010, J Biol Chem285(46):35740-9; Golubkov et al., 2012, J Biol Chem 287(50):42009-18; Naet al., 2012, J Biol Chem 287(30):25001-9). Circulating antigen couldimpact the pharmacokinetics of therapeutic compounds such as a PTK7 ADC.Circulating levels of shed PTK7 were evaluated from various serumsources. PTK7 protein levels were measured with a quantitative assayusing the Meso Scale Discovery (MSD®) platform.

Serum samples from healthy humans were purchased from the StanfordUniversity Blood Bank. Serum samples from cancer patients were purchasedfrom Asterand Inc and Bioreclamation Inc. Cynomolgus monkey serumsamples were purchased from Bioreclamation Inc.

Mouse serum samples were obtained from immune-compromised mice thatharbored human tumor xenografts. Female non-obese diabetic-severecombined immunodeficiency (NOD-scid) mice were purchased from HarlanLaboratories® and housed in accordance with Institutional Animal Careand Use Committee (IACUC) guidelines. Patient-derived xenografts (PDX)were established by direct implantation of freshly resected human tumorsamples and propagated by passaging xenografts into naïve animals. Thexenografts were derived from primary tumor resection samples that wereprocured from clinical sites following Institutional Review Board forthe Protection of Human Subjects approval and in accordance with HealthInsurance Portability and Accountability Act (HIPAA) regulations.

The assay to measure levels of PTK7 protein utilized two specificanti-PTK7 monoclonal antibodies (mAbs) that had been generated byhybridoma technology. The mAbs bind human and cynomolgus monkey PTK7 butnot murine PTK7. The assay was developed on the MSD® platform and wasoptimized for a linear response. A MSD® high bind plate was coated withPTK7-specific mAb H2.35 at 1 g/ml in phosphate-buffered saline (PBS).The plate was incubated at 4° C. overnight. The next day the plate waswashed and the second mAb was added. For human and monkey samples, 25 μlof sulfo-tagged PTK7-specific mAb 6M38 was added at 0.5 g/ml in MSD®diluent2 (MSD #R51 BB-4), and for tumor-bearing mouse samples,biotinylated 6M38 was added at 0.5 g/ml in MSD® Diluent 2 (MSD #R51BB-4) followed by horseradish peroxidase-conjugated streptavidin. Theplate was incubated with shaking for 30 minutes. After the incubation,without washing, serum samples or varying amounts of recombinant PTK7protein were added to the wells and incubated for 2 hours on a plateshaker. The serum samples were diluted 4× to 25% final in MSD® Diluent2. The plates were washed 3 times with phosphate-buffered saline with0.2% Tween-20. MSD® 1× Read buffer (MSD #R92TC-3) was added to theplates (150 μl per well), and the plates were read on the MSD® SectorImager. Values for serum samples were interpolated from the standardcurve based on recombinant protein.

The levels of PTK7 protein in human serum were measured in samples fromhealthy humans and cancer patients that represented 8 tumor types. Theresults are summarized in Table 10. The reported value in each categoryindicates the mean of all individual samples. The mean value of PTK7 inserum from healthy humans was 12.4±3.3 ng/mL. In general, the meanvalues for cancer patients were slightly higher, ranging up to 24.6±3.8ng/mL and with a broader distribution of individual values (FIG. 6).

TABLE 10 PTK7 Protein Levels in Human Serum Number of PTK7 ProteinSample Type Samples Level (ng/mL) Healthy human 30 12.4 ± 3.3  Breastcancer patients 29 24.0 ± 24.1 Colorectal cancer patients 17 13.3 ± 4.3 Melanoma patients 6 15.4 ± 7.4  Non-small cell lung cancer 21 19.8 ±12.1 patients Ovarian cancer patients 7 17.9 ± 8.1  Pancreatic cancerpatients 9 20.4 ± 6.5  Prostate cancer patients 14 16.2 ± 5.7  Smallcell lung cancer patients 7 24.6 ± 3.8  ng/mL = nanograms permilliliter. Measurements are provided as Mean ± Standard Deviation ofthe Mean for the independent biological samples.

The levels of PTK7 protein in naïve cynomolgus monkey serum weremeasured in samples from 29 animals. The mean value was 35.8±13.4 ng/mL(Table 11) which is higher than the corresponding values for healthyhumans and cancer patients.

TABLE 11 PTK7 Protein Levels in Cynomolgus Monkey Serum Number PTK7Protein Level Sample Type of samples (ng/mL) Healthy cynomolgus 29 35.8± 13.4 monkey ng/mL = nanograms per milliliter. Measurement is providedas Mean ± Standard Deviation of the Mean for the independent biologicalsamples.

Mouse serum samples were obtained from immune-compromised mice thatharbored human tumor xenografts. Specifically, the xenografts were PDXswhich typically preserve the architecture and genotype of the humantumors from which they are derived (DeRose et al, 2011, Nat Med17(11):1514-20). The mean values of PTK7 protein in serum for all 11tumor types were <1 ng/mL for all tumor types (Table 12) and, thus,significantly lower than the values obtained for human and monkey. Sincethe mAbs used in the assay do not cross-react with murine PTK7, thevalues are interpreted as human PTK7 protein that was shed from thetumor xenografts and not normal murine tissues.

TABLE 12 PTK7 Protein Levels in Tumor-Bearing Mouse Serum Number of PTK7Protein Tumor Xenograft Tumor Models Level (ng/mL) Naïve (no xenograft)Not applicable 0 ± 0 Breast cancer 11 0.454 ± 0.872 Colorectal cancer 290.023 ± 0.083 Head and neck cancer 2 0.355 ± 0.501 Kidney cancer 7 0.004± 0.009 Liver cancer 7 0.008 ± 0.021 Non-small cell lung cancer 20 0.065± 0.104 Ovarian cancer 9 0.053 ± 0.086 Pancreatic cancer 9 0.018 ± 0.055Prostate cancer 2 0 ± 0 Skin cancer 9 0.208 ± 0.307 Small cell lungcancer 10 0.004 ± 0.011 ng/mL = nanograms per milliliter. Measurementsare provided as Mean ± Standard Deviation of the Mean for the tumormodels. Values for individual models were the median of measurementsfrom 1 to 12 tumor-bearing animals.

Example 7 Internalization

Antibody internalization is a critical characteristic for deliveringADCs for cytotoxicity in PTK7 expressing cells. Anti-PTK7 antibody hu24was observed to internalize into cancer cells, which suggests that theantibody is a suitable vehicle for delivering a toxin into the cells.Adherent cells were dissociated using TrypLE Express (Gibco),neutralized with cell culture media and then counted. Cells werealiquoted into a U-bottom 96 well plate with 5×10⁵ cells/100 μL mediaper well. The plate was centrifuged at 300×g, for 5 minutes at 4° C. topellet the cells and the supernatants were aspirated. All reagents werekept on ice for the following steps.

Each cell pellet was resuspended in 3 μg/ml hu24 or non-binding antibody(Human IgG, Thermo Scientific) in 100 μL 3% BSA in PBS. The plate wasincubated on ice for 30 minutes and then centrifuged, and the cellpellets were washed in 200 μL 3% BSA in PBS. The cell pellets wereresuspended in 100 μL 37° C. pre-warmed cell culture media and placed ina 37° C. incubator for 1 or 4 hours. The cell pellets to be incubated at4° C. were similarly resuspended and then placed on ice. After theincubations, samples were centrifuged, supernatants aspirated and washedwith 200 L/well ice cold 3% BSA in PBS and resuspended in 100 μL/wellice-cold 3% BSA in PBS and placed on ice. All samples are thencentrifuged, supernatants were aspirated, each cell pellet wasresuspended in 100 μL of Allophycocyanin (APC)-conjugated anti-Human IgGFc fragment that had been diluted 1:50 in ice-cold 3% BSA in PBS. Theplate was incubated on ice for 30 minutes and then centrifuged, and thecell pellets were washed in 200 μL 3% BSA in PBS, resuspended in 100 μL3% BSA in PBS, and transferred to a 5 mL polycarbonate tube containing250 μL 3% BSA in PBS. The samples were analyzed by flow cytometry using5 μL 7-AAD per sample as a viability stain. The mean fluorescentintensity (MFI) was measured for each sample with non-viable cellsexcluded from the analysis. The value for “% internalized” wascalculated as (100%−[MFI after incubation/MFI before incubation]). Theresults in Table 13 indicate that the hu24 antibody was internalizedinto all the cell lines tested, and that the internalization wastemperature-dependent, thus reflecting active (not passive)internalization by the cell.

TABLE 13 % internalization (relative to start of experiment) Number ofCell line 1 hr at 37° C. 4 hrs at 37° C. 1 hr at 4° C. 4 hrs at 4° C.experiments BT549 14.3 ± 9.8  42.5 ± 9.1 4.3 ± 8.1 8.5 ± 7.7 6 H661 21.2± 12.7   36 ± 17.4  2.2 ± 11.5   2 ± 5.6 5 MDAMB468 21.7 ± 5.7  34.3 ±9.0 7.0 ± 0.8 9.3 ± 2.5 3

Example 8 Cytotoxicity Mediated by Saporin-Conjugated Anti-Human FabFragment

An in vitro cytotoxicity assay was performed to determine whether hu23or hu24 antibody can mediate the delivery of a cytotoxic agent to celllines. In this respect, anti-human IgG Fab fragment covalently linked tothe saporin toxin (Advanced Targeting Systems) was combined withunlabeled hu23, hu24 or 8.84 Ab (non-binding, negative control antibody)and then incubated with cells for 4 days (PTK7 expressing cells H446 andDMS114) or 7 days (OE19 non-PTK7 expressing cells; OE21 PTK7 expressingcells) after which cell viability was measured.

In one experiment, H446 or DMS114 cancer cell lines were plated into aclear flat-bottom tissue culture plate at 9600 cells per well (H446) or6400 cells per well (DMS114) in 100 μl of cell culture media. The cellswere incubated overnight at 37° C. in a 5% CO₂ incubator. On thefollowing day, 50 μl of hu23, hu24 or 8.84 Ab pre-mixed withsaporin-conjugated anti-human IgG Fab (Fab-ZAP; Advanced TargetingSystems) at 1:2 molar ratio was added to the cells on a 10-pointconcentration curve with triplicate samples starting with 1 μg/ml with1:3 dilutions in cell culture media. The plate was incubated in a 37°C., 5% CO₂ incubator for 4 days. To measure cell viability, the MTSassay (Promega Cell Titer 96 Aqueous Non-Radioactive Cell ProliferationAssay) was used according to the supplier's instructions. 30 μL of thecombined MTS reagent was added to each well. The plate was incubated ina 37° C., 5% CO₂ incubator for 2 hours. The Optical Density (OD) wasdetermined at 490 nm with a 96-well plate reader. The average readingfrom wells with media alone was subtracted from the readings of wellswith cells to control for background OD. The data was subjected tologistic non-linear regression analysis (GraphPad Prism Software) inorder to determine the concentration of primary antibody at which cellviability was inhibited by 50% (IC50).

The data in Table 14 indicates that both anti-PTK7 antibodies hu23 andhu24 conferred saporin-mediated cytotoxicity to the H446 and DMS114cells, while the 8.84 negative control antibody did not. The resultsdemonstrate that the activity of hu23 and hu24 was specific for PTK7expressing cells.

TABLE 14 IC₅₀ Values (ng/mL) Cell Line hu23 + Fab-ZAP hu24 + Fab-ZAP8.84 + Fab-ZAP H446 44.7 60.3 >1000 DMS114 10.3 12.0 >1000

In another experiment, the saporin assay was performed on two cell linesderived from esophageal cancers, OE19 and OE21 (Sigma Aldrich). Todetermine PTK7 expression on the cell lines, the cells were cultured andsingle cell suspensions were isolated using Versene (Invitrogen). Cellswere washed in PBS/2% FCS and incubated with hu24 antibody or HuIgG1(isotype control) at a concentration of 5 μg/mL for 30 minutes. Cellswere washed again in PBS/2% FCS, then incubated at 1:200 with anti-humanAlexa Fluor647 (Jackson Immunoresearch) for 20 minutes. Cells werewashed again, resuspended in DAPI, and then analyzed on a BD FACSCantoto determine the change in mean fluorescence intensity (ΔMFI). OE19cells did not exhibit staining fluorescence above the isotype control(ΔMFI=0) whereas the OE21 cells exhibited almost a two-log increase influorescence intensity (ΔMFI=5976), which indicated the expression ofPTK7 on the surface of OE21, an esophageal squamous cell carcinoma.

To determine whether hu24 can mediate the delivery of cytotoxic agents,2500 cells/well of a dissociated single cell suspension from either OE21or OE19 were plated on BD Tissue Culture plates (BD Biosciences) inculture medium. One day after plating, various concentrations ofpurified hu24 and a fixed concentration of 4 nM anti-HuIgG Fab fragmentcovalently linked to saporin (Advanced Targeting Systems) were added tothe cultures. After a 7-day incubation, viable cell numbers wereenumerated using CELL TITER GLO® (Promega) as per manufacturer'sinstructions. Raw luminescence counts using cultures containing cellswith the saporin Fab fragment were set as 100% reference values and allother counts calculated accordingly (referred to as “Normalized RLU”).Using this assay it was demonstrated that hu24 mediated cytotoxicityagainst OE21 cells, but not OE19 cells, and the isotype control did notaffect cell counts, as shown in Table 15. These results indicate thatcell binding of the antibody hu24 is required to elicit thesaporin-mediated cytotoxicity to PTK7 expressing cell but has no effecton a non-PTK7 expressing cell.

TABLE 15 IC50 Values (μg/ml) Cell Line hu24 + Fab-ZAP HulgG1 control +Fab-ZAP OE21 0.5 >100 OE19 >100 >100

Example 9 Synthesis of vc0101 and mc8261

The synthesis of vc0101 (vc is the linker and 0101 is the drug) andmc8261 (mc is the linker and 8261 is the drug) was prepared according tothe methods described in International Publication No. WO/2013/072813,which is herein incorporated by reference in its entirety.

A. Experimental Method for the Synthesis of vc0101

Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[(21S,24S,25R)-24-[(2S)-butan-2-yl]-25-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-18,18,23-trimethyl-3,16,19,22-tetraoxo-21-(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N˜5˜-carbamoyl-L-ornithinamide(vc0101).

Step 1. Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#53). To a solution of compound #32 (2.05 g, 2.83 mmol, 1 eq.) indichloromethane (20 mL, 0.1 M) and N,N-dimethylformamide (3 mL) wasadded the amine #19 (2.5 g, 3.4 mmol, 1.2 eq.), HATU (1.29 g, 3.38 mmol,1.2 eq.) and triethylamine (1.57 mL, 11.3 mmol, 4 eq.). The mixture wasstirred at room temperature while reaction progress was monitored byLC-MS and TLC. Once complete, the reaction was concentrated in vacuo,the residue was azeotroped three times with heptanes, and the resultingcrude product was purified by silica gel chromatography (Gradient: 0% to55% acetone in heptane), producing compound #53 (2.42 g, 74%) as asolid. LC-MS: m/z 965.7 [M+H⁺], 987.6 [M+Na⁺], retention time=1.04minutes (Protocol H-below); HPLC (Protocol A-below): m/z 965.4 [M+H⁺],retention time=11.344 minutes (purity >97%); ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: δ7.86-7.91 (m, 2H), [7.77 (d, J=3.3 Hz) and 7.79 (d, J=3.2 Hz), total1H], 7.67-7.74 (m, 2H), [7.63 (d, J=3.2 Hz) and 7.65 (d, J=3.2 Hz),total 1H], 7.38-7.44 (m, 2H), 7.30-7.36 (m, 2H), 7.11-7.30 (m, 5H),[5.39 (ddd, J=11.4, 8.4, 4.1 Hz) and 5.52 (ddd, J=11.7, 8.8, 4.2 Hz),total 1H], [4.49 (dd, J=8.6, 7.6 Hz) and 4.59 (dd, J=8.6, 6.8 Hz), total1H], 3.13, 3.17, 3.18 and 3.24 (4 s, total 6H), 2.90 and 3.00 (2 br s,total 3H), 1.31 and 1.36 (2 br s, total 6H), [1.05 (d, J=6.7 Hz) and1.09 (d, J=6.7 Hz), total 3H].

Step 2. Synthesis of 0101:2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#54).

To a solution of compound #53 (701 mg, 0.726 mmol) in dichloromethane(10 mL, 0.07 M) was added diethylamine (10 mL), and the reaction mixturewas stirred at room temperature while reaction progress was monitored byLC-MS and TLC. Once complete, the reaction was concentrated in vacuo,the residue was azeotroped three times with heptanes, and the resultingcrude product was purified by silica gel chromatography (Gradient: 0% to10% methanol in dichloromethane). The residue was diluted with diethylether and heptane and was concentrated in vacuo to afford #54 (406 mg,75%) as a white solid. LC-MS: m/z 743.6 [M+H⁺], retention time=0.70minutes (Protocol F-below); HPLC (Protocol A-below): m/z 743.4 [M+H⁺],retention time=6.903 minutes, (purity >97%); ¹H NMR (400 MHz, DMSO-d₆),presumed to be a mixture of rotamers, characteristic signals: 6 [8.64(br d, J=8.5 Hz) and 8.86 (br d, J=8.7 Hz), total 1H], [8.04 (br d,J=9.3 Hz) and 8.08 (br d, J=9.3 Hz), total 1H], [7.77 (d, J=3.3 Hz) and7.80 (d, J=3.2 Hz), total 1H], [7.63 (d, J=3.3 Hz) and 7.66 (d, J=3.2Hz), total 1H], 7.13-7.31 (m, 5H), [5.39 (ddd, J=11, 8.5, 4 Hz) and 5.53(ddd, J=12, 9, 4 Hz), total 1H], [4.49 (dd, J=9, 8 Hz) and 4.60 (dd,J=9, 7 Hz), total 1H], 3.16, 3.20, 3.21 and 3.25 (4 s, total 6H), 2.93and 3.02 (2 br s, total 3H), 1.21 (s, 3H), 1.13 and 1.13 (2 s, total3H), [1.05 (d, J=6.7 Hz) and 1.10 (d, J=6.7 Hz), total 3H], 0.73-0.80(m, 3H).

Step 3. Synthesis of vc0101:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[(21S,24S,25R)-24-[(2S)-butan-2-yl]-25-(2-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-2-oxoethyl)-18,18,23-trimethyl-3,16,19,22-tetraoxo-21-(propan-2-yl)-2,7,10,13,26-pentaoxa-4,17,20,23-tetraazaheptacos-1-yl]phenyl}-N˜5˜-carbamoyl-L-ornithinamide.

The coupling of compound 0101 (#54) to linker vc(N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide)(MalcValCitPABC-PNP) was accomplished according to General Procedure E(below) using appropriate quantities of DMA as the solvent, and HOAT and2,6-Lutidine as additives, and the resulting crude desired material waspurified according the Method D (below) to give 33 mg (36%) of thedesired product. Under conditions specified in Protocol A (below) withthe column maintained at 45° C., this material gave an HPLC retentiontime of 9.114 minutes (Protocol A-below); LC-MS: m/z 1342.6 [M+H⁺],retention time 3.48 minutes (Protocol H-below).

B. Experimental Method for the Synthesis of mc8261

Preparation ofN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(mc8261).

Step 1. Synthesis of methylN-{(2R3R)-3-methoxy-2-methyl-3-[(2S)-pyrrolidin-2-yl]propanoyl}-L-phenylalaninate,hydrochloride salt (#67). According to General Procedure C (below), from#37 (2.39 g, 5.33 mmol, 1 eq.), dioxane (10 mL, 0.53 M) and a 4Mhydrochloric acid solution in dioxane (10 mL, 40 mmol, 7.5 eq.) wassynthesized #67 (2.21 g) as a white solid, which was used in the nextstep without further purification. LC-MS: m/z 349.2 [M+H⁺], retentiontime=0.53 minutes; ¹H NMR (400 MHz, DMSO-d₆) δ9.45-9.58 (br m, 1H), 8.63(d, J=8.1 Hz, 1H), 8.51-8.62 (br m, 1H), 7.25-7.33 (m, 4H), 7.18-7.25(m, 1H), 4.50 (ddd, J=10.8, 8.1, 4.5 Hz, 1H), 3.65 (s, 3H), 3.54 (dd,J=6.8, 4.5 Hz, 1H), 3.20 (s, 3H), 3.11 (dd, J=13.8, 4.5 Hz, 1H),2.99-3.14 (br m, 3H), 2.89 (dd, J=13.8, 10.9 Hz, 1H), 2.44-2.50 (m, 1H,assumed; partially obscured by solvent peak), 1.77-1.89 (m, 1H),1.60-1.73 (m, 2H), 1.46-1.57 (m, 1H), 1.05 (d, J=6.8 Hz, 3H).

Step 2. Synthesis ofN-[(9H-fluoren-9-ylmethoxy)carbonyl]-2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide(#68). According to General Procedure D (below), from #32 (353 mg, 0.488mmol, 1 eq.),

in dichloromethane (10 mL, 0.04 M), amine #67 (271 mg, 50.588 mmol, 1.3eq.), HATU (223 mg, 0.586 mmol, 1.2 eq.) and diisopropylethylamine (238μL, 1.71 mmol, 3.5 eq.) was synthesized the crude desired material,which was purified by silica gel chromatography (Gradient: 0% to 40%acetone in heptane), affording #68 (404 mg, 88% over two steps) as asolid. LC-MS: m/z 940.7 [M+H⁺], 962.7 [M+Na⁺], retention time=1.04minutes; HPLC (Protocol C-below): retention time=9.022 minutes; ¹H NMR(400 MHz, DMSO-d₆), presumed to be a mixture of rotamers, characteristicsignals: 8 [8.25 (br d, J=8 Hz) and 8.48 (br d, J=8 Hz), total 1H], 7.89(d, J=7.4 Hz, 2H), 7.67-7.75 (m, 2H), 7.38-7.44 (m, 2H), 7.31-7.36 (m,2H), 7.14-7.24 (m, 5H), 4.43-4.69 (m, 3H), 4.17-4.26 (m, 3H), 3.91-3.99(br m, 1H), 3.63 and 3.65 (2 s, total 3H), 3.19 and 3.24 (2 s, total3H), 3.14 and 3.15 (2 s, total 3H), 2.90 and 2.99 (2 br s, total 3H),1.36 and 1.37 (2 br s, total 3H), 1.30 and 1.32 (2 s, total 3H), [1.02(d, J=6.8 Hz) and 1.06 (d, J=6.6 Hz), total 3H].

Step 3A. Synthesis of 8261:2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide,trifluoroacetic acid salt (#69).

To a solution of #68 (143 mg, 0.152 mmol, 1 eq.) in tetrahydrofuran (5mL, 0.02 M) was added a solution of lithium hydroxide (9.10 mg, 0.378mmol, 2.5 eq.) in water (3 mL). After 5 hours, the reaction wasconcentrated in vacuo, azeotroped three times with heptane, dissolved indimethyl sulfoxide (2.2 mL) and purified by reverse phase chromatography(Method C-below) to give #69 (56 mg, 52%). HPLC (Protocol A-below at 45°C.): 704.4 [M+H⁺], retention time=6.623 minutes; ¹H NMR (400 MHz,DMSO-d₆), presumed to be a mixture of rotamers, characteristic signals:δ 8.08-8.22 and 8.37-8.49 (2 m, total 5H), 7.12-7.28 (m, 5H), 3.18, 3.20and 3.24 (3 s, total 6H), 2.95 and 3.04 (2 br s, total 3H), 1.52 and1.53 (2 s, total 3H), 1.39 and 1.41 (2 s, total 3H), [1.02 (d, J=6.8 Hz)and 1.05 (d, J=6.6 Hz), total 3H], 0.74-0.81 (m, 3H).

Step 4: Synthesis of mc8261:N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-2-methylalanyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide.

The coupling of compound 8261 (#69) to linker maleimidocaproyl (mc):

was accomplished according to General Procedure D (below) and theresulting crude desired material was purified according the Method C(below) to give 30.2 mg (24%) of the desired product. Under conditionsspecified in Protocol A (below) with the column maintained at 45° C.,this material gave an HPLC retention time of 9.058 minutes (ProtocolA-below); LC-MS: m/z 897.7 [M+H⁺], retention time 0.81 minutes (ProtocolH-below).

C. General Procedures, Methods and Protocols

General Procedure C:

Boc removal or tert-butyl ester (also refers to t-Bu ester) cleavageusing hydrochloric acid in dioxane. To either a solution ofBoc-containing compound or tert-butyl ester-containing compound indioxane (or in some cases no solution, or other relevant solvent) wasadded a 4 M solution of hydrochloric acid in dioxane. Reaction progresswas monitored by LC-MS (or HPLC or TLC). The reaction was concentratedin vacuo and in some cases azeotroped one to four times with heptanes.

General Procedure D:

coupling with O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU). To a stirring solution of the amine (1.0eq.) and acid (1.0-2.0 eq.) in dichloromethane, N,N-dimethylformamide(also referred to as DMF), or a mixture of both, HATU (1.0-2.0 eq.) wasadded followed by triethylamine (2.0-4.0 eq.) or diisopropylethylamine(2.0-4.0 eq., also referred to as Hunig's base). Reaction progress wasmonitored by LC-MS (or HPLC or TLC); the reaction was usually completedwithin three hours. Solvents were removed in vacuo. The residue waspurified by silica gel or reverse phase chromatography or in some casesazeotroped three times with heptanes, diluted with a small amount ofethyl acetate before being reduced down onto silica or C18 bonded silicaand purified by silica gel or reverse phase chromatography.

General Procedure E:

coupling withN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(MalcValCitPABC-PNP). To a mixture of the payload amine (1 eq.) andN-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N⁵-carbamoyl-N-[4-({[(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide(MalcValCitPABC-PNP, Eur. Pat. Appl. (1994), EP624377, 1.0-2.0 eq.) inN,N-dimethylformamide or dimethylacetamide (also referred to as DMA),pyridine (0.0-4.0 eq.), diisopropylethylamine (0.0-4.0 eq.),2,6-dimethylpyridine (0.0-4.0 eq., also referred to as 2,6-Luditine) and1-hydroxybenzotriazole hydrate (0.01-1.1 eq. also referred to as HOBT)or 3H-[1,2,3]triazolo[4,5-b]pyridin-3-ol (0.01-1.1 eq., also referred toas HOAT) was added. After stirring at 40° C.-50° C. for 1-48 hours, thereaction mixture was concentrated in vacuo and azeotroped three timeswith heptane. The crude material was purified by reverse phasechromatography according to the specified method to afford the desiredmaterial.

Method C:

Column: Phenomenex Luna C18, 100×30 mm, 10 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in methanol (v/v); Gradient: 10% to 90% B over 20minutes; Flow rate: 20 mL/minute. Temperature: not controlled;Detection: DAD 210 nm, 254 nm; Injection Volume: variable; Instrument:Gilson.

Method D:

Column: Phenomenex Synergi Max-RP, 150×21. 2 mm, 4 pm; Mobile phase A:0. 1% formic acid in water; Mobile phase B: 0. 1% formic acid inacetonitrile; Gradient: 30% B for 1. 5 minutes, 30% to 60% B over 8. 5minutes, 60 to 100% B over 0. 5 minutes then 100% B over 2 minutes; Flowrate: 27 mL/minute; Detection: DAD 210-360 nm; MS (+) range 150-20005daltons; Instrument: Waters FractionLynx.

Protocol A:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.1%formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 1.5 minutes, 5% to 100% B over8.5 minutes, then 100% B for 1 minute; Flow rate: 0.75 mL/minute.Temperature: 25° C.; Detection: DAD 215 nm; MS (+) range 150-2000daltons; Injection volume: 10 μL Instrument: Agilent 1200 LCMS.

Protocol C:

Column: Phenomenex Luna C18 (2), 150×3.0 mm, 5 μm; Mobile phase A: 0.02%trifluoroacetic acid in water (v/v); Mobile phase B: 0.02%trifluoroacetic acid in methanol (v/v); Gradient: 50% to 100% B over 10minutes; Flow rate: 0.75 mL/minute. Temperature: not controlled;Detection: DAD 215 nm, 254 nm; Injection volume: 10 μL; Instrument:Agilent 1100 HPLC.

Protocol F:

Column: Waters Acquity UPLC BEH, C18, 2.1×50 mm, 1.7 μm; Mobile phase A:0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 5% B over 0.1 minute, 5% to 95% B over 0.7minute, 95% B over 0.1 minute; Flow rate: 1.25 mL/minute. Temperature:60° C.; Detection: 200-450 nm; MS (+) range 100-1200 daltons; Injectionvolume: 5 μL; Instrument: Waters Acquity.

Protocol H:

Column: Phenomenex Gemini-NX, C18, 4.6×50 mm, 3 μm, 110 Å; Mobile phaseA: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid inacetonitrile (v/v); Gradient: 0% to 100% B over 4.10 minutes, linearthen 100% B over 0.4 minute; Flow rate: 1.5 mL/minute. Temperature: 60°C.; Detection: DAD 200-450 nm; MS (+) range 100-2000 daltons; Injectionvolume: 5 μL; Instrument: Agilent.

Example 10 Bioconjugation of the Anti-PTK7 Antibodies

A. Anti-PTK7-vc0101 Antibody-Drug Conjugates

In the present invention, anti-PTK7 antibodies hu23, hu24 and hu58 wereconjugated to vc0101 to generate hu23-vc0101 ADC, hu24-vc0101 ADC andhu58-vc0101 ADC, or conjugated to mc8261 to generate hu23-mc8261 ADC,hu24-mc8261 ADC and hu58-mc8261 ADC. The conjugation of hu23, hu24, andhu58 to vc0101 mc8261 was achieved by derivatizations of the side chainsof cysteine residues. These cysteines are normally paired as inter-chaincysteine disulfide bridges, of which there are 4 conserved pairs(involving 8 cysteine residues) on an IgG1 antibody. Partial reductionof these disulfide linkages provides a distribution of free thiols thatcan be functionalized with the maleimide handle on the vc linker.Specifically, an anti-PTK7 antibodies of the present invention werepartially reduced via addition of 2.4 molar excess oftris(2-carboxyethyl)phosphine (TCEP) in 100 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), pH 7.0 and1 mM diethylenetriaminepentaacetic acid (DTPA) for 2 hours at 37° C. Thevc0101 or mc8 261 linker-payload was then added to the reaction mixtureat a linker-payload/antibody molar ratio of 7 and reacted for anadditional 1 hour at 25° C. in the presence of 15% v/v ofdimethylacetamide (DMA). After the 1 hour incubation period, 3-foldexcess of N-ethylmaleimide was added to cap the unreacted thiols and wasallowed to react for 15 minutes, followed by addition of 6-fold excessL-Cys to quench any unreacted linker-payload.

The reaction mixture was dialyzed overnight at 4° C. in phosphatebuffered saline (PBS), pH 7.4, and purified via size exclusionchromatography (SEC; AKTA explorer, Superdex 200). The final ADC drugsubstance was formulated in 20 mM Histidine, 85 mg/mL Sucrose, pH 5.8buffer.

The anti-PTK antibody-drug conjugates were further characterized via SECfor purity and hydrophobic interaction chromatography (HIC), and liquidchromatography electrospray ionization mass spectrometry (LC-ESI MS)which was used to calculate drug-antibody ratio (drug loading). Theprotein concentration was determined via ultraviolet (UV)spectrophotometry. This method provides an antibody-drug conjugate as aheterogeneous mixture of functionalized antibodies that contain anaverage drug-to-antibody ratio (DAR) of approximately 4 mol/mol.

The drug distribution profile was assessed via HIC for hu24-vc0101 andpresented in the chromatogram in FIG. 7. Briefly, analytical HIC wasperformed on TSK gel butyl-NPR column. The ADC was bound to the columnin 1.5 M ammonium sulfate, 50 mM potassium phosphate dibasic, pH 7 andeluted with 50 mM potassium phosphate dibasic and 20% isopropanol (IPA),pH 7.

B. Anti-PTK7-AcBut CM Antibody-Drug Conjugates

In the present invention, anti-PTK7 antibodies hu23, hu24 and hu58 wereconjugated to AcBut-N-acetyl-γ-calicheamicin dimethyl hydrazide(AcButCM) OSu ester to generate hu23-AcButCM ADC, hu24-AcButCM ADC andhu58-AcButCM ADC as shown below, wherein X can be any antibody, such ashu23, hu24 and hu58.

The reaction mixture included 10 mg/ml or less anti-PTK7 antibody andAcButCM OSu ester at a molar ratio of 4-4.5 to 1. High agitation wasconducted during the addition of AcButCM to a mixing vortex. Thereaction pH was 8.3 and the concentrations of other reaction componentswere as follows: 180 mM HEPES buffer, 41 mM sodium decanoate, and 8%(v/v) ethanol. The reaction was conducted at 33° C. for 5 minutes. Afterthe conjugation reaction was completed, the reaction mixture was dilutedslowly with 1.3 volumes of 1 M K₂HPO₄ adjusted to pH 8.5 with mixing.

To purify, the diluted above reaction mixture was loaded in two batcheson a Butyl Sepharose-4 Fast Flow HIC column (GE Healthcare) that waspreviously equilibrated in five column volumes (cv) of 0.52M potassiumphosphate buffer, pH 8.5. The protein loaded on the column was 3.5 mg/mlof bed volume. The flow rate was 15 ml/minute through the sample loadingand 22 ml/minute throughout the wash and elution phase of thechromatography. This improved gradient removes higher DAR ADCs that werebound to the column.

The unbound fraction during the loading was predominantly reactionreagents and most of the unconjugated antibody, which was discarded. Thecolumn was then washed with 0.3 cv of 0.52M potassium phosphate buffer,pH 8.5, to remove any remaining reagents. A step gradient with 1 cv from0.52M to 0.4M potassium phosphate buffer, pH 8.5 was then used to eluteany loosely bound unconjugated antibody along with low loadedanti-PTK7-AcButCM, if present. The main fraction was then eluted using astep gradient of 1 cv from 0.4M to 5 mM potassium phosphate buffer, pH8.5, to provide anti-PTK7-AcButCM having a DAR in the range of 3 to 5,toward the end of the gradient. If anti-PTK7-AcButCM conjugates with ahigher DAR were present, the fraction was eluted using a gradient of 2cv of 5 mM potassium phosphate buffer, pH 8.5, and then an elution ofpure deionized water. Any anti-PTK7-AcButCM conjugates with a higher DARthat remained bound after the deionized water elution were eluted using2 cv of 10 mM sodium hydroxide containing 20% ethanol. The purifiedbatches contained anti-PTK7-AcButCM conjugates with a DAR of 3 to 5.

This improved conjugation and purification processes generated ADCshaving a DAR that was less than 6, and in some aspects in the range of 3to 5. Further, the processes generated a narrower distribution ofloading, for example, less heterogeneity within the product.Improvements to the conjugation and purification processes furtherincluded: 1) decreasing the AcButCM to anti-PTK7 antibody ratio to 4-4.5to 1 to generate an ADC having a lower DAR, 2) conducting high agitationduring addition of AcButCM to anti-PTK7 antibody to generate ADCs withlow amounts of unconjugated antibody (free antibody), 3) reducingincubation time to 5 minutes, compared to 60-90 minutes, to provide lowaggregates and 4) a reduction in ethanol amount to 6-8% to provide lowaggregates. The purified pooled peaks from both batches were dialyzedtwice against a formulated buffer to facilitate storage in a frozenstate. The formulated buffer composition was 20 mM Tris, 7.5% sucrose,0.01% polysorbate 80, 10 mM NaCl, pH 8.0.

Example 11 Binding Characteristics of Hu24 mAb and Hu24 ADC

To determine whether hu24 mAb and hu24-vc0101 ADC bind to the cynomolgusmonkey ortholog of PTK7, the cynomologus monkey PTK7 protein was clonedand expressed. Sequence analysis revealed that the protein is 97.9%identical to the human PTK7 protein.

Surface plasmon resonance (SPR) analysis was conducted to characterizethe binding characteristics of the mAb and ADC to human and cynomolgusmonkey PTK7 protein ectodomains. Binding was determined to be comparableby SPR analysis. There was no significant difference in affinitiesbetween mAb and ADC for human or cynomolgus monkey PTK7 protein (Table16). All K_(on) measurements are within 3-fold and all K_(off)measurements are within 2-fold, ranges considered in the field to bewithin typical systematic error and therefore not likely to bephysiologically significant.

TABLE 16 Affinity Constants of hu24 and hu24-vc0101 for Human PTK7 andCynomolgus Monkey PTK7 Test Article Antigen Species K_(on) (M⁻¹s⁻¹)K_(off) (s⁻¹) Kd (nM) hu24 mAb Human 7.9E+05 5.4E−04 0.7 Cynomolgus4.3E+05 5.2E−04 1.2 hu24-vc0101 Human 5.1E+05 5.3E−04 1.4 Cynomolgus2.8E+05 1.0E−03 3.6 K_(on) = Association rate constant; K_(off) =Dissociation rate constant; Kd = Equilibrium dissociation constant; M =Molar; mAb = Monoclonal antibody; nM = Nanomolar; s = Seconds

The ability of anti-PTK7 mAbs to bind to PTK7 orthologs was alsoevaluated by sandwich ELISA. Briefly, human, cynomolgus monkey, rat ormouse PTK7-His tagged protein was captured on an ELISA plate by directcoating. All antigens were captured at 1 μg/ml, 100l/well. Hu24 mAb andhu24-vc0101 ADC were serially diluted starting with 810 ng/ml and addedto the washed and blocked wells to test for binding to the antigen. mAband ADC binding was detected by polyclonal anti-human IgG horseradishperoxidase conjugate and read out with TMB substrate.

Both mAb and ADC bound comparably to human and cynomolgus monkey PTK7proteins by ELISA (Table 17). Together with the SPR results, theseresults confirmed cross-reactivity to cynomolgus monkey PTK7 anddemonstrated that the bioconjugation process did not change of theobserved binding characteristics of the mAb. However, neither mAb norADC exhibited detectable binding to rat or mouse PTK7 protein at100-fold higher antigen concentration than needed to observe binding tohuman PTK7 (Table 17). Rat and mouse antigens were confirmed to becorrectly folded by binding to known cross reactive antibodies (data notshown).

TABLE 17 Binding of hu24 and hu24-vc0101 to PTK7 proteins by ELISA ED50values (ng/mL) Cynomolgus Human PTK7 PTK7 Rat PTK7 Mouse PTK7 hu24 mAb4.7 5.0 No binding No binding hu24-vc0101 6.6 7.4 No binding No bindingADC = Antibody-drug conjugate; ED50 = Effective dose that gives 50%maximum signal; mAb = Monoclonal antibody; ng/mL = Nanograms permilliliter

Unconjugated hu24 and hu24 conjugated to vc0101 were compared for theirability to bind to PTK7 expressing cells by flow cytometry using themethods described in Example 3. Briefly, cultured H1975 or EKVX cellswere harvested and incubated at 4° C. with hu24-vc0101 ADC orunconjugated hu24 mAb followed by fluorophore-conjugated secondary mAband a viability stain and then analyzed by flow cytometry.

Table 18 provides the mean channel fluorescence of the viable cellpopulation. Comparable data was obtained in an independent replicateexperiment thus conjugation of hu24 to the linker payload does not alterits binding to PTK7 expressing cells.

TABLE 18 Comparable Cell Binding of Conjugated and Unconjugated hu24Concentration of hu24- Cell vc0101 ADC or hu24 mAb Line Test Article 0.1μg/mL 0.3 μg/mL 1 μg/mL 3 μg/mL H1975 hu24-vc0101 ADC 94 196 302 341hu24 mAb 91 183 310 348 EKVX hu24-vc0101 ADC 4 4 4 5 hu24 mAb 4 4 4 5

Example 12 In Vitro Cytotoxicity Assays

The cytotoxicity of the antibody-drug conjugate hu24-vc0101 wasevaluated on cell lines that express the target PTK7. The engineeredHEK293T-PTK7 over-expressing cell line was plated into a clearflat-bottom tissue culture plate (BD Falcon) at 500 cells per 180 μL ofcell culture media per well. Human cancer cell lines were also tested inthis assay and plated at a density that was previously determined to beoptimal for each cell line according to their rate of growth (H661 1500cells/well and H446 9600 cells/well in 150 μl). The cells were incubatedovernight at 37° C. in a 5% CO₂ incubator. On the following day, thehu24-vc0101 ADC and the 8.84 Ab-vc0101 ADC (negative control) were addedto the cells on a 10 point concentration curve in triplicate samplesstarting with 3 or 10 μg/mL with 1:3 dilutions in cell culture media.The plate was incubated in a 37° C., 5% CO₂ incubator for 4 days. TheMTS assay (Promega CellTiter 96 Aqueous Non-Radioactive CellProliferation Assay) was used according to the supplier's instructions.30 μL (H446, H661) or 40 μL (HEK293T-PTK7) of the combined MTS reagentwas added to each well, and the plate was incubated in a 37° C., 5% CO₂incubator for 2 hours. The OD was determined at 490 nm with a 96-wellplate reader. The average reading from wells with media alone wassubtracted from the readings of wells with cells to control forbackground OD. The data was subjected to logistic non-linear regressionanalysis (GraphPad Prism Software) in order to determine theconcentration of antibody-drug conjugate at which cell viability wasinhibited by 50% (IC50).

Table 19 provides IC50 values for the hu24-vc0101 ADC in thecytotoxicity assay. In all three cell lines, hu24-vc0101 elicited potentcytotoxicity whereas the non-binding 8.84 Ab-vc0101 did not; these datademonstrate that the potent cytotoxicity of hu24-vc0101 was dependent onanti-PTK7 antibody.

TABLE 19 IC50 Values (ng/mL) Cell Line hu24-vc0101 ADC 8.84 Ab-vc0101ADC Free 0101 HEK293-PTK7 1.7 >3000 ND H661 27.5 ± 20.5 >10000 0.33 H4467.6 ± 5.0 >10000 0.59 ND = not determined

Example 13 In Vivo Efficacy of Anti-PTK7 Antibody-Drug Conjugates

The effects of anti-PTK7 antibody-drug conjugates were further evaluatedon the in vivo growth of human tumor patient-derived xenografts (PDX).Primary tumor resection samples were procured from clinical sitesfollowing Institutional Review Board for the Protection of HumanSubjects approval and in accordance with HIPAA regulations. Tumorfragments were stored and shipped in Hypothermasol (Biolife Solutions)on ice and were embedded in Matrigel (BD) containing a proprietary mixof growth factors and implanted subcutaneously into the mammary fatpadof female NOD/SCID mice within 24 hours of resection. Mice weremonitored for health status daily and for tumor growth initially byvisual inspection twice per week. Once the tumors were palpable,measurements of tumor volume began to track tumor growth and estimatecell doubling time. Tumor volume was estimated using the equationV=(A*B²)/2 where A is the long axis and B is the short axis. When tumorsreached a volume of 500 mm³ to 1,500 mm³, they were harvested for studyand for re-transplant as a patient-derived xenograft (PDX). Depending onthe line, mechanical and/or chemical dissociation can be used toseparate the individual cells for passage. Live cells were inoculatedinto naïve animals with 10,000 to 50,000 cells per animals

For efficacy studies, tumors were harvested from passaging studies andcells were dissociated into single cell suspension. Preparations werecounted for live cells using Trypan blue exclusion and 10,000 to 50,000cells were inoculated per mouse in Matrigel. To account for differentialgrowth rates of PDX, at least 25% more animals were started to allow forminimal tumor volume variance at randomization. Tumor growth wasinitially followed by palpability with measurements beginning once tumorvolumes reached about 30 mm³. Studies were randomized based on tumorsize once a cohort of tumor-bearing mice reached 140 mm³ to 180 mm³.Animals were dosed by intraperitoneal injection twice a week for twoweeks (q4d×4). Study groups were followed until individual mice orentire group tumor measurements reached 1200 mm³ when sacrifice wasindicated in accordance with IACUC protocol. For selected dosingstudies, pharmacokinetic submandibular bleeds were performed at 2 hours,36 hours and 72 hours. A volume of 10 μL of blood was immediatelypipetted into 90 μL of HBS-P (GE Healthcare). Samples were stored at −8°C. prior to analysis. For each tumor measurement the tumorvolume±standard error of the mean (SEM) is provided. GT=Group Terminateddue to large tumor size. All studies included a control antibody drugconjugate comprised of a non-binding hIgG1 antibody conjugated to thesame linker-payload being analyzed and with comparable drug-to-antibodyratio (DAR) and loading distribution.

Tables 20-37 demonstrate the effectiveness of the antibody-drugconjugates hu23-vc0101, hu24-vc0101,hu58-vc0101, hu23-AcButCM,hu24-AcButCM and hu58-AcButCM in PDX models established using varioushuman tumor cells that have a relative PTK7 expression determined to below, medium, or high.

A. Breast Cancer (BR)

Table 20 and FIG. 8 show that the hu23-vc0101, hu24-vc0101, andhu58-vc0101 ADCs are all effective in a PDX model using the humanBreast-13 (BR13) triple-negative breast cancer (TNBC) PDX model (highPTK7 expression) compared to vehicle and drug controls. The hu24-vc0101ADC was more effective than both hu23-vc0101 and hu58-vc0101 in the BR13PDX model. All of the PTK7 ADCs tested were more effective thandoxorubicin, which is a standard of care treatment for TNBC.

TABLE 20 Efficacy of anti-PTK7-vc0101 ADCs in BR13 TNBC PDX. 1 mg/kg 3mg/kg 3 mg/kg 3 mg/kg 3 mg/kg Day Vehicle Doxorubicin Control-vc0101hu23-vc0101 hu24-vc0101 hu58-vc0101 0 144 ± 13 156 ± 13 153 ± 9  154 ±9  153 ± 10  151 ± 10 7 174 ± 13 219 ± 29 211 ± 17 199 ± 12 194 ± 12 183 ± 14 14 255 ± 22 295 ± 39 277 ± 28 206 ± 16 175 ± 17  159 ± 15 21349 ± 40 353 ± 45 323 ± 44 116 ± 16 97 ± 11 110 ± 12 28 428 ± 48 432 ±50 413 ± 48 48 ± 7 41 ± 4   61 ± 13 35 734 ± 74 696 ± 84 537 ± 66 39 ± 637 ± 5  37 ± 3 42  940 ± 105 1001 ± 159 591 ± 74 23 ± 7 20 ± 5  27 ± 349 GT GT 775 ± 87 23 ± 7 19 ± 7  40 ± 5 56 GT GT 952 ± 89 15 ± 6 4 ± 432 ± 3 63 GT GT 1181 ± 119  6 ± 4 0 ± 0 40 ± 6 70 GT GT 1287 ± 98   7 ±5 0 ± 0 48 ± 7 77 GT GT 1449 ± 125  8 ± 5 0 ± 0  64 ± 10 84 GT GT GT  14± 10 0 ± 0  85 ± 15 91 GT GT GT  20 ± 11 0 ± 0 119 ± 28 98 GT GT GT  39± 18 0 ± 0 165 ± 26 105 GT GT GT  52 ± 22 0 ± 0 236 ± 29 112 GT GT GT 94 ± 41 0 ± 0 314 ± 37 119 GT GT GT 116 ± 50 0 ± 0 389 ± 51 126 GT GTGT 123 ± 52 0 ± 0 538 ± 73 133 GT GT GT 152 ± 71 0 ± 0 624 ± 76 140 GTGT GT 204 ± 97 0 ± 0 849 ± 78 147 GT GT GT  257 ± 125 0 ± 0 935 ± 74 154GT GT GT  318 ± 141 0 ± 0 954 ± 65 161 GT GT GT  352 ± 148 0 ± 0 GT

FIGS. 9 and 10 show the efficacy of the hu23-mc8261, hu24-mc8261, andhu58-mc8261 ADCs in the BR13 TNBC PDX model (high PTK7 expression) incomparison to hu24-vc0101. Treatment with hu24-vc0101 yielded sustainedtumor regression for over 200 days and demonstrated greater inhibitionof tumor growth compared to anti-PTK7-mc8261 ADCs.

Table 21 and FIG. 11 show that the hu23-vc0101, hu24-vc0101, andhu58-vc0101 ADCs are all effective in a PDX model using the humanBreast-22 (BR22) TNBC PDX model (high PTK7 expression) compared tovehicle and drug controls. In this model the hu23-vc0101 and hu24-vc0101ADCs were more effective than hu58-vc0101, demonstrating similarantibodies to the same target (PTK7) have varying degrees of efficacy.All of the PTK7 ADCs tested were more effective than docetaxel, which isa standard of care treatment for TNBC. Notably, docetaxel and the drugcomponent of the ADCs, auristatin 0101, have similar mechanisms ofaction in that they inhibit tubulin polymerization.

TABLE 21 Efficacy of anti-PTK7-vc0101 ADCs in BR22 TNBC PDX. 3 mg/kg 1mg/kg 3 mg/kg 3 mg/kg 3 mg/kg 20 mg/kg Control- hu24- hu24- hu58- hu23-Day Vehicle Docetaxel vc0101 vc0101 vc0101 vc0101 vc0101 0 171 ± 19 182± 21 164 ± 14 172 ± 22  170 ± 19  143 ± 7  150 ± 9  7 354 ± 52 154 ± 23221 ± 34 245 ± 28  89 ± 22 169 ± 13  100 ± 11  14 655 ± 99 107 ± 51 207± 49 157 ± 48  21 ± 5  49 ± 12 10 ± 6  21 1028 ± 142 218 ± 98 129 ± 40213 ± 97  0 ± 0 38 ± 24 1 ± 1 28 GT  418 ± 165 209 ± 71 266 ± 114 0 ± 084 ± 55 0 ± 0 35 GT 1191 ± 264  482 ± 142 357 ± 156 0 ± 0 157 ± 91  0 ±0 42 GT GT  724 ± 212 758 ± 225 0 ± 0 301 ± 178 0 ± 0 49 GT GT 1054 ±308 912 ± 203 0 ± 0 584 ± 339 0 ± 0 56 GT GT GT GT 0 ± 0 813 ± 381 0 ± 063 GT GT GT GT 0 ± 0 943 ± 409 0 ± 0 70 GT GT GT GT 0 ± 0 GT 0 ± 0 77 GTGT GT GT 0 ± 0 GT 0 ± 0 84 GT GT GT GT 0 ± 0 GT 0 ± 0 91 GT GT GT GT 0 ±0 GT 0 ± 0 98 GT GT GT GT 0 ± 0 GT 0 ± 0 105 GT GT GT GT 0 ± 0 GT 0 ± 0112 GT GT GT GT 0 ± 0 GT 0 ± 0 119 GT GT GT GT 0 ± 0 GT 0 ± 0 126 GT GTGT GT 0 ± 0 GT 0 ± 0 133 GT GT GT GT 0 ± 0 GT 0 ± 0 140 GT GT GT GT 0 ±0 GT 0 ± 0 147 GT GT GT GT 0 ± 0 GT 0 ± 0 154 GT GT GT GT 0 ± 0 GT 0 ± 0161 GT GT GT GT 0 ± 0 GT 0 ± 0

Table 22 shows the hu23-AcButCM, hu24-AcButCM, and hu58-AcButCM ADCswere all effective in the BR22 TNBC PDX model (high PTK7 expression)compared to vehicle and drug controls. However, hu58-AcButCM was moreeffective than both hu23-AcButCM and hu24-AcButCM, illustrating theunpredictable nature of the use of various payloads with similarantibodies to the same target. The hu23-vc0101 and hu24-vc0101 ADCs weremore effective than hu58-vc0101, whereas the hu58-AcButCM was moreeffective than both hu23-AcButCM and hu24-AcButCM ADCs. All of the PTK7ADCs tested were more effective than doxorubicin, which is a standard ofcare treatment for TNBC.

TABLE 22 Efficacy of ADCs in BR22 TNBC PDX. 0.3 mg/kg 0.3 mg/kg 0.3mg/kg 0.3 mg/kg 1.5 mg/kg Control- hu23- hu58- hu24- Day VehicleDoxorubicin AcButCM AcButCM AcButCM AcButCM 0 303 ± 16 297 ± 17 186 ± 16186 ± 15 157 ± 13  193 ± 16  14 797 ± 71 641 ± 39 456 ± 55 140 ± 15 30 ±5  185 ± 71  28 GT GT 1019 ± 148 11 ± 2 0 ± 0 32 ± 28 63 GT GT GT 100 ±83 10 ± 10 118 ± 117 91 GT GT GT  652 ± 479 67 ± 67 723 ± 695 119 GT GTGT  999 ± 527 125 ± 125 862 ± 797 133 GT GT GT GT 125 ± 125 968 ± 794161 GT GT GT GT 125 ± 125 1538 ± 1098 175 GT GT GT GT 150 ± 150 GT

FIGS. 12 and 13 show the efficacy of the hu23-mc8261, hu24-mc8261, andhu58-mc8261 ADCs in the BR22 TNBC PDX model (high PTK7 expression) incomparison to hu23-vc0101. Treatment with hu23-vc0101 yielded sustainedtumor regressions for over 200 days and demonstrated greater inhibitionof tumor growth as compared to anti-PTK7-mc8261 ADCs.

Tables 23-25 and FIG. 14 show the efficacy of the hu23-vc0101,hu24-vc0101, hu58-vc0101, hu23-mc8261 and hu24-mc8261, hu58-mc8261 andhu23-AcButCM, hu24-AcButCM, hu58-AcButCM ADCs in the Breast-31 (BR31)TNBC PDX model (high PTK7 expression). In this model, all three ADCShaving vc0101 linker-payload were more effective than the ADCs havingAcButCM or mc8261. This result was unexpected since the AcButCM linkerpayload was generally more potent than the vc0101 linker payload in thein vivo PDX models tested. In addition, the PTK7-vc0101 ADCs were moreeffective than docetaxel and the PTK7-AcButCM ADCs were more effectivethan doxorubicin. Both docetaxel and doxorubicin are standards of carefor TNBC.

TABLE 23 Efficacy of anti-PTK7-vc0101 ADCs in BR31 TNBC PDX. 20 mg/kg 3mg/kg 3 mg/kg 3 mg/kg 3 mg/kg Day Vehicle Docetaxel Control-vc0101hu23-vc0101 hu24-vc0101 hu58-vc0101 0 159 ± 10 259 ± 47 148 ± 12 164 ±14  159 ± 15  158 ± 12  7 269 ± 17 331 ± 70 227 ± 23 178 ± 23  188 ± 12 170 ± 25  14 425 ± 32 371 ± 82 133 ± 10 79 ± 8  82 ± 7  100 ± 11  21 668± 50 451 ± 91 101 ± 21 28 ± 7  21 ± 3  38 ± 3  28 1088 ± 93   510 ± 103 70 ± 17 8 ± 3 0 ± 0 21 ± 7  35 GT  614 ± 117 105 ± 28 0 ± 0 0 ± 0 8 ± 542 GT  790 ± 117  21 ± 76 0 ± 0 0 ± 0 0 ± 0 49 GT  837 ± 126 234 ± 83 2± 0 0 ± 0 30 ± 18 56 GT ND  393 ± 103 2 ± 1 0 ± 0 3 ± 1 63 GT 1131 ± 148 680 ± 181 2 ± 1 0 ± 0 17 ± 9  70 GT 1317 ± 182 1169 ± 300 8 ± 3 0 ± 035 ± 18 77 GT GT 1300 ± 331 13 ± 4  1 ± 1 70 ± 24 84 GT GT GT 32 ± 10 2± 2 108 ± 32  91 GT GT GT 59 ± 24 10 ± 10 143 ± 36  98 GT GT GT 59 ± 1513 ± 13 213 ± 46  105 GT GT GT 115 ± 35  111 ± 92  295 ± 68  112 GT GTGT 249 ± 75  370 ± 269 551 ± 155 119 GT GT GT 293 ± 72  521 ± 429 691 ±143 126 GT GT GT 449 ± 100 551 ± 428 826 ± 155 133 GT GT GT 541 ± 135745 ± 499 914 ± 155 140 GT GT GT 658 ± 130 765 ± 494 GT 147 GT GT GT 810± 145 794 ± 488 GT

TABLE 24 Efficacy of anti-PTK7-mc8261 ADCs in BR31 TNBC PDX. 10 mg/kg 10mg/kg 10 mg/kg Day Vehicle hu23-mc8261 hu24-mc8261 hu58-mc8261 0  159 ±10 154 ± 8  154 ± 9  161 ± 13 7  269 ± 17 238 ± 7  202 ± 20 225 ± 21 14 425 ± 32 304 ± 12 275 ± 35 280 ± 29 21  668 ± 50 383 ± 32 370 ± 53 390± 39 28 1088 ± 93 601 ± 70 609 ± 81 635 ± 55 35 GT 857 ± 88  862 ± 139 889 ± 118

TABLE 25 Efficacy of anti-PTK7-AcButCM ADCs in BR31 TNBC PDX. 0.3 mg/kg0.3 mg/kg 0.3 mg/kg 0.3 mg/kg 1.5 mg/kg Control- hu58- hu23- hu24- DayVehicle Doxorubicin AcButCM AcButCM AcButCM AcButCM 0 159 ± 10 160 ± 11145 ± 11  145 ± 12 144 ± 9  149 ± 11 7 269 ± 17 304 ± 20 223 ± 25  199 ±21 236 ± 20 188 ± 14 28 1088 ± 93   759 ± 111 675 ± 158 270 ± 42 236 ±36 106 ± 7  35 GT GT 869 ± 204 381 ± 46 339 ± 69 139 ± 15 63 GT GT GT 885 ± 150  920 ± 208 299 ± 41 77 GT GT GT GT GT  590 ± 104 84 GT GT GTGT GT  882 ± 183

Table 26 and FIG. 15 show the hu24-vc0101 ADC was effective in the humanBreast-64 (BR64) TNBC PDX model (medium PTK7 expression) compared tovehicle and drug controls. The data demonstrates that a PDX model havinga moderate expression of the PTK7 target was targeted effectively withhu24-vc0101. Hu24-vc0101 was more effective than docetaxel, a standardof care for TNBC.

TABLE 26 Efficacy of hu24-vc0-10-1 in BR64 TNBC PDX. 20 mg/kg 3 mg/kg 3mg/kg Day Vehicle Docetaxel Control-vc0101 hu24-vc0101 0 499 ± 36 543 ±46 190 ± 30 173 ± 24  7  832 ± 119 1112 ± 97  418 ± 62 271 ± 30  14 1768± 284 1805 ± 206 839 ± 91 180 ± 55  21 GT GT 1907 ± 576 96 ± 30 28 GT GTGT 53 ± 21 35 GT GT GT 15 ± 6  42 GT GT GT 27 ± 22 49 GT GT GT 65 ± 5856 GT GT GT 144 ± 141 63 GT GT GT 394 ± 392 70 GT GT GT 422 ± 385 77 GTGT GT 690 ± 460 84 GT GT GT 1094 ± 594 

FIG. 16 shows the efficacy of the hu24-vc0101, and the lack of efficacyof the unconjugated hu24, in the BR5 TNBC PDX model. Mice harboring Br5human breast cancer xenografts were dosed every four days for fourcycles (Q4D×4) with hu24-vc0101 ADC, unconjugated hu24 mAb or vehicle.Tumor measurements were recorded twice a week using digital calipers andare shown as Mean±SEM. The 3 mg/kg dose of hu24-vc0101 ADC caused tumorregression without tumor growth through day 49. In contrast, theunconjugated hu24 mAb did not inhibit tumor growth (FIG. 16). Thus theobserved efficacy is dependent on delivery of the linker-payload.

FIG. 17 shows the efficacy of the hu24-vc0101 in comparison topaclitaxel in the BR36 PR+TNBC PDX model. Mice harboring BR36 humanbreast cancer xenografts were dosed Q4D×4 with hu24-vc0101 ADC,paclitaxel, negative control ADC or vehicle. Tumor measurements wererecorded twice a week using digital calipers and are shown as Mean±SEM.The 3 mg/kg dose of hu24-vc0101 ADC caused tumor regression withouttumor growth through day 103. Hu24-vc0101 out preformed paclitaxeladministered at the MTD. The negative control ADC exhibited only modestactivity (FIG. 17).

B. Small Cell Lung Cancer (SCLC)

The data in Tables 27-28 illustrate the effectiveness of the hu24-vc0101and hu24-AcButCM ADCs in the small cell lung cancer-64 (LU64) PDX model(low PTK7 expression). In this model, both ADCs were shown to beeffective in this PDX of low PTK7 expression although the ADCs having anAcButCM linker payload were more effective than ADCs having a vc0101linker payload. Both hu24-vc0101 and hu24-AcButCM were more effectivethan the standard of care for SCLC (which is cisplatin plus etoposide).

TABLE 27 Efficacy of hu24-vc0101 ADCs in LU64 PDX. 5 mg/kg Cisplatin + 3mg/kg 10 mg/kg 24 mg/kg Control- Control- 3 mg/kg 10 mg/kg Day VehicleEtoposide vc0101 vc0101 hu24-vc0101 hu24-vc0101 0 139 ± 12 139 ± 10 156± 11 168 ± 14 166 ± 15 178 ± 19 7 241 ± 25 25 ± 5 255 ± 26 312 ± 38 252± 11 348 ± 32 14 376 ± 33 31 ± 5 379 ± 26 433 ± 49 310 ± 38 391 ± 42 21613 ± 39  77 ± 22 493 ± 38 619 ± 72 389 ± 35 377 ± 66 28 1087 ± 86  151± 38 718 ± 92 824 ± 85 537 ± 67 500 ± 76 35 GT 214 ± 54  912 ± 158 GT651 ± 70  618 ± 113 42 GT 285 ± 72 GT GT  932 ± 128  773 ± 141 49 GT 738 ± 127 GT GT GT  838 ± 171 56 GT 1011 ± 172 GT GT GT 1054 ± 185 63GT GT GT GT GT 1367 ± 290

TABLE 28 Efficacy of hu24-AcButCM ADCs in LU64 PDX. 5 mg/kg Cisplatin +0.1 mg/kg 0.02 mg/kg 0.05 mg/kg 0.1 mg/kg 24 mg/kg Control- hu24- hu24-hu24- Day Vehicle Etoposide AcButCM AcButCM AcButCM AcButCM 0 139 ± 12139 ± 10 171 ± 11 137 ± 10 137 ± 10  136 ± 9  21 613 ± 39  77 ± 22 575 ±45 13 ± 4 1 ± 1 0 ± 0 28 1087 ± 86  151 ± 38 723 ± 77 12 ± 4 0 ± 0 0 ± 035 GT 214 ± 54  998 ± 107  24 ± 10 0 ± 0 0 ± 0 42 GT 285 ± 72 GT  36 ±17 0 ± 0 0 ± 0 56 GT 1011 ± 172 GT 168 ± 55 0 ± 0 0 ± 0 63 GT GT GT 274± 89 0 ± 0 0 ± 0 105 GT GT GT  760 ± 206 0 ± 0 0 ± 0 112 GT GT GT GT 0 ±0 0 ± 0 168 GT GT GT GT 0 ± 0 0 ± 0

Table 29 shows the efficacy of the hu23-mc8261, hu24-mc8261, andhu58-mc8261 ADCs in the LU64 PDX model (low PTK7 expression). Treatmentwith hu24-vc0101 demonstrated greater inhibition of tumor growth thanthe anti-mc8261 ADCs in this model.

TABLE 29 Efficacy of anti-PTK7-mc8261 ADCs in LU64 PDX. 10 mg/kg 10mg/kg 10 mg/kg Day Vehicle hu23-mc8261 hu24-mc8261 hu58-mc8261 0 184 ±24 171 ± 11 170 ± 12 177 ± 15 7 269 ± 26 239 ± 14 257 ± 29 279 ± 32 14417 ± 16 311 ± 19 330 ± 45 364 ± 31 21 591 ± 37 455 ± 38 435 ± 76 517 ±47 28  738 ± 103 572 ± 51  622 ± 119 760 ± 74 35 771 ± 92 770 ± 92  903± 197 1029 ± 95  42 970 ± 64 1088 ± 116 GT GT

Tables 30-31 show the efficacy of the hu24-vc0101, hu23-vc0101 andhu24-AcButCM ADCs in the small cell lung cancer-86 (LU86) PDX model(high PTK7 expression). In this model, both hu24-vc0101 and hu23-vc0101were effective in suppressing tumor growth. Hu24-vc0101 was moreeffective than the control-vc0101 ADC. However, the hu24-AcButCM ADC wasmore potent than both of the ADCs having a vc0101 linker payload (Table22) providing further example of the general greater potency of AcButCMcompared to vc0101 in SCLC models. Hu24-AcButCM was more effective thancisplatin plus etoposide, which is a standard of care for SCLC.

TABLE 30 Efficacy of anti-PTK7-vc0101 ADCs in LU86 PDX. 3 mg/kg 0.3mg/kg 1 mg/kg 3 mg/kg 3 mg/kg Control- hu24- hu24- hu24- hu23- DayVehicle vc0101 vc0101 vc0101 vc0101 vc0101 0 139 ± 11 164 ± 19 145 ± 11143 ± 12  141 ± 11  200 ± 23  7 334 ± 44 228 ± 45 362 ± 39 282 ± 46  194± 24  42 ± 11 14  758 ± 100 310 ± 39 758 ± 62 363 ± 77  56 ± 11 0 ± 0 211213 ± 140 394 ± 57 1441 ± 97  420 ± 115 5 ± 4 0 ± 0 28 GT  748 ± 102 GT621 ± 159 0 ± 0 0 ± 0 35 GT 1279 ± 198 GT 784 ± 141 0 ± 0 0 ± 0 42 GT GTGT 915 ± 105 0 ± 0 0 ± 0 49 GT GT GT GT 0 ± 0 0 ± 0 56 GT GT GT GT 0 ± 00 ± 0 63 GT GT GT GT 17 ± 17 0 ± 0 70 GT GT GT GT 73 ± 45 0 ± 0 77 GT GTGT GT 121 ± 67  0 ± 0 84 GT GT GT GT 204 ± 90  0 ± 0 91 GT GT GT GT 477± 272 0 ± 0 98 GT GT GT GT 570 ± 270 0 ± 0 105 GT GT GT GT 689 ± 281 19± 19 112 GT GT GT GT GT 72 ± 72 119 GT GT GT GT GT 154 ± 154 203 GT GTGT GT GT 154 ± 154

TABLE 31 Efficacy of hu24-AcButCM ADCs in LU86 PDX. 5 mg/kg Cisplatin +0.3 mg/kg 0.03 mg/kg 0.1 mg/kg 0.3 mg/kg 24 mg/kg Control- hu24- hu24-hu24- Day Vehicle Etoposide AcButCM AcButCM AcButCM AcButCM 0 206 ± 25149 ± 13 158 ± 20  147 ± 13 143 ± 12  150 ± 13  14 1019 ± 6  468 ± 47393 ± 115 756 ± 79 443 ± 90  73 ± 25 21 GT 946 ± 77 571 ± 173 1245 ± 138548 ± 120 18 ± 5  28 GT GT 788 ± 166 GT 761 ± 154 3 ± 2 35 GT GT 962 ±176 GT 908 ± 155 0 ± 0 42 GT GT GT GT GT 0 ± 0 112 GT GT GT GT GT 0 ± 0140 GT GT GT GT GT 0 ± 0 154 GT GT GT GT GT 0 ± 0 203 GT GT GT GT GT 0 ±0

Table 32 shows the efficacy of the hu23-mc8261, hu24-mc8261, andhu58-mc8261 ADCs in the LU86 PDX model (high PTK7 expression). Treatmentwith hu23-vc0101 yielded sustained tumor regression in the LU86 PDXmodel and demonstrated greater inhibition of tumor growth as compared toanti-PTK7-mc8261 ADCs

TABLE 32 Efficacy of anti-PTK7-mc8261 ADCs in LU86 PDX. 10 mg/kg 10mg/kg 10 mg/kg Day Vehicle hu23-mc8261 hu24-mc8261 hu58-mc8261 0 147 ±11 159 ± 13 159 ± 14 158 ± 15 7 317 ± 33 228 ± 29 257 ± 27 225 ± 20 14672 ± 62 256 ± 30 300 ± 37 321 ± 34 21 1233 ± 83  340 ± 48 383 ± 65 425± 42 28 GT 455 ± 68 544 ± 90 574 ± 57 35 GT 740 ± 78  815 ± 117 736 ± 7642 GT 870 ± 99  903 ± 157  887 ± 106 49 GT 1165 ± 137 1333 ± 173 1265 ±206

FIGS. 18A-B show the efficacy of PTK7 ADCs conjugated to either 0101 orCM in two different SCLC PDX models, a H1048 PDX model (high PTK7expression) and SCLC 95 PDX model (high PTK7 expression), respectively

FIG. 19A-B shows the efficacy of hu24-AcBut CM in two different SCLC PDXmodels, a SCLC 117 PDX model (moderate PTK7 expression) and a SCLC 102PDX model (low PTK7 expression), respectively. The results demonstratethat hu24-AcButCM or hu23-AcButCM ADCs are more effective thanhu24-vc0101 ADCs against SCLC. This finding is unexpected in light ofthe strong anti-tumor activity of hu24-vc0101 in PDX models of othertumor types such as TNBC and NSCLC. In addition, the results suggestthat activity of the ADC correlates with expression of PTK7, sincehu24-AcButCM ADC elicited the weakest response in SCLC102 which has lowPTK7 expression.

C. Non-Small Cell Lung Cancer (NSCLC)

Table 33 and FIG. 20 show the hu24-vc0101 ADC was effective in the humannon-small cell lung cancer-135 (LU135) PDX model (high PTK7 expression)compared to vehicle and drug controls. This data demonstrates theeffectiveness of hu24-vc0101 in suppressing tumor growth in a NSCLC PDX.Hu24-vc0101 was more effective than paclitaxel, which is a standard ofcare in NSCLC.

TABLE 33 Efficacy of hu24-vc0101 in LU135 PDX. 20 mg/kg 1 mg/kg 3 mg/kg1 mg/kg 3 mg/kg Day Vehicle Paclitaxel Control-vc0101 Control-vc0101hu24-vc0101 hu24-vc0101 0 162 ± 17 175 ± 24  161 ± 15 168 ± 18 158 ± 18186 ± 23 7 446 ± 28 77 ± 13 461 ± 19 329 ± 39 369 ± 53 79 ± 6 14 615 ±34 58 ± 12 655 ± 39 337 ± 50 520 ± 81 41 ± 4 21 830 ± 71 60 ± 11 752 ±46 414 ± 31 651 ± 95 60 ± 8 28 982 ± 68 93 ± 20  815 ± 101 488 ± 40  838± 133  84 ± 15 35 GT 110 ± 31  GT 627 ± 60 GT 104 ± 17 42 GT 184 ± 48 GT  871 ± 117 GT 175 ± 26 49 GT 284 ± 78  GT GT GT 244 ± 19 56 GT 384 ±106 GT GT GT 347 ± 34 63 GT 538 ± 178 GT GT GT 387 ± 47 70 GT 667 ± 174GT GT GT 502 ± 96 77 GT 724 ± 171 GT GT GT  628 ± 152

Table 34 and FIG. 21 show the hu24-vc0101 ADC was effective in the humannon-small cell lung cancer-176 (LU176) PDX model (high PTK7 expression)compared to vehicle and drug controls. This data demonstrates theeffectiveness of hu24-vc0101 in suppressing tumor growth in a NSCLC PDX.Hu24-vc0101 was more effective than cisplatin, which is a standard ofcare in NSCLC.

TABLE 34 Efficacy of hu24-vc0101 in LU176 PDX. 3 mg/kg 1 mg/kg 3 mg/kg 5mg/kg Control- hu24- hu24- Day Vehicle Cisplatin vc0101 vc0101 vc0101 0262 ± 17 259 ± 26 139 ± 9  148 ± 12 138 ± 10 7 658 ± 71 124 ± 30 245 ±29  446 ± 222  77 ± 21 14  964 ± 196  97 ± 22 208 ± 26  401 ± 214 27 ± 621 2087 ± 381 102 ± 24 244 ± 54  458 ± 204 34 ± 9 28 GT 179 ± 71 366 ±88  685 ± 193 21 ± 3 35 GT 207 ± 69  679 ± 154 1046 ± 288 22 ± 8 42 GT297 ± 97  955 ± 129 GT 20 ± 7 49 GT  619 ± 262 GT GT  28 ± 14 56 GT GTGT GT 14 ± 4 63 GT GT GT GT 10 ± 4 70 GT GT GT GT  5 ± 2 77 GT GT GT GT 9 ± 5 84 GT GT GT GT  8 ± 8 91 GT GT GT GT  11 ± 11 98 GT GT GT GT  21± 16 105 GT GT GT GT  28 ± 27 112 GT GT GT GT  46 ± 44 119 GT GT GT GT 140 ± 140 126 GT GT GT GT  281 ± 281 133 GT GT GT GT  366 ± 366 140 GTGT GT GT  649 ± 649

D. Ovarian Cancer (OV)

Table 35 shows the efficacy of the hu24-vc0101 and hu24-AcButCM ADCs inthe ovarian-55 (OV55) PDX model (medium PTK7 expression) compared tovehicle and drug controls. This data demonstrates that a ovarian PDXmodel that has a moderate expression of the PTK7 target is effectivelytargeted by both ADCs. Surprisingly, the animals treated withhu24-vc0101 were tumor free when the experiment was terminated and thehu24-AcButCM ADC was less effective in this model.

TABLE 35 Efficacy of hu24 ADCs in OV55 PDX. 0.1 mg/kg 3 mg/kg 0.1 mg/kg3 mg/kg Day Control-AcButCM Control-vc0101 hu24-AcButCM hu24-vc0101 0168 ± 7  167 ± 7  162 ± 6  166 ± 8  14 438 ± 58 134 ± 46 105 ± 15 33 ±6  35 1204 ± 46   427 ± 196  137 ± 101 0 ± 0 42 GT  564 ± 210  194 ± 1580 ± 0 49 GT  807 ± 265  294 ± 204 0 ± 0 77 GT GT  895 ± 169 0 ± 0 84 GTGT GT 0 ± 0 133 GT GT GT 0 ± 0 168 GT GT GT 0 ± 0 182 GT GT GT 0 ± 0

E. Melanoma (SK)

Table 36 shows the hu24-vc0101 ADC was effective in the humanmelanoma-23 (SK23) PDX model (medium PTK7 expression) compared tovehicle and drug controls. This data demonstrates the effectiveness ofhu24-vc0101 in a melanoma PDX model having a moderate expression of thePTK7 target, providing a potential indication for the use of the ADC.

TABLE 36 Efficacy of hu24-vc010-1 in SK23 PDX. 2 mg/kg 4 mg/kg 2 mg/kg 4mg/kg Day Control-vc0101 Control-vc0101 hu24-vc0101 hu24-vc0101 0 183 ±20 184 ± 14 187 ± 11 196 ± 17 7 587 ± 82 541 ± 54 432 ± 28 356 ± 45 14 871 ± 107 697 ± 61 456 ± 36 227 ± 45 21 1123 ± 86  910 ± 93 509 ± 51150 ± 33 28 GT GT 679 ± 65 135 ± 41 35 GT GT 793 ± 65 166 ± 58 42 GT GT868 ± 63 178 ± 62 49 GT GT GT 228 ± 81 56 GT GT GT 250 ± 93 63 GT GT GT 295 ± 103 70 GT GT GT  398 ± 130 77 GT GT GT  568 ± 181 84 GT GT GT 688 ± 167

F. Tumor Growth Inhibition in Breast and NSCLC PDX Models

Animals were dosed every four days for four cycles (Q4D×4) byintraperitoneal injection except the BR5 study which was by intravenousinjection on the same schedule. Tumor measurements were recorded one ortwo times per week using digital calipers and tumor volume was estimatedusing the equation V=(A×B²)/2 where A is the long axis and B is theshort axis. Animal body weights were measured and recorded at least oncea week. Study groups were followed until individual mice or entire grouptumor measurements reached 1200 mm³ at which point sacrifice wasindicated in accordance with IACUC protocol. For the BR5 study, animalswere sacrificed once tumor volume approached 15% of the body weight atstaging in accordance with IACUC protocol.

Tumor growth inhibition (TGI) was calculated using the equation %TGI=[1−(mean tumor volume of treated)/(mean tumor volume of vehicle)].TGI was calculated at the latest possible time point, which wastypically the last measurement before the control group was discontinuedas described above. Tumor regression was defined as a reduction in meantumor volume after dosing. In cases where tumors regressed, Time ToProgression (TTP) indicates the number of days between the first doesand the time at which mean tumor volume increased from the previousmeasurement to a statistically significant degree. If the tumor did notregrow during the course of the experiment, TTP is the number of daysbetween the first dose and the end of the experiment.

To confirm exposure of hu24-vc0101 ADC in the efficacy studies, plasmaconcentrations of ADC and total antibody were determined for two PDXmodels, BR13 PDX and BR22 PDX. Samples were collected at three timepoints following the third administration of ADC and concentrations weremeasured by Ligand Binding Assay (LBA) (data not shown). The dataindicate that drug exposures were comparable in these tumor models.

Hu24-vc0101 ADC elicited anti-tumor activity in breast cancer and NSCLCtumor models. Tumors regressed upon treatment and typically did notregrow for months after the last administration of ADC. The unconjugatedhu24 mAb did not elicit anti-tumor activity in the model tested whichdemonstrated the auristatin-dependent mechanism of action. Results aresummarized in Table 37.

TABLE 37 Summary of In Vivo Pharmacology Studies with hu24-vc0101 ADCTarget hu24-vc0101 Expression Dosing Exposure Dose Level Regression(TTP, Tumor Model by IHC Regimen Data (mg/kg) Days) or % TGI BR13 TNBCPDX High Q4Dx4 ip Yes 1 37% TGI 2 Regression (35)  4 Regression (105)BR22 TNBC PDX High Q4Dx4 ip Yes 0.3 None 1 Regression (21)  3 Regression(205) BR31 TNBC Moderate Q4Dx4 ip No 3 Regression (105) PDX BR5 TNBC PDXHigh Q4Dx4 iv No 3 Regression (49)  hu24 mAb, None 3 mg/kg BR36 PR+ PDXHigh Q4Dx4 ip No 3 Regression (103) NSCLC135 PDX High Q4Dx4 ip No 1 15%TGI 3 Regression (32)  NSCLC176 PDX High Q4Dx4 ip No 1 78% TGI 3Regression (98)  % = percent; BR = Breast Cancer; IHC =Immunohistochemistry; ip = Intraperitoneal; iv = Intravenous; mAb =Monoclonal antibody; mg/kg = Milligram per kilogram; NSCLC = Non-SmallCell Lung Cancer; PDX = Patient-Derived Xenograft; PR+ = ProgesteroneReceptor Positive; Q4Dx4 = Dose Every 4 Days for 4 Cycles; TGI = TumorGrowth Inhibition; TNBC = Triple-Negative Breast Cancer; TTP = Time ToProgression.

Example 14 Mechanism of Action of 0101

To study the mechanism of action of the hu24-vc0101 ADC, cells weretreated with the ADC and then their microtubule structure assessed.Auristatin is a fully synthetic dolastatin-based pentapeptide inhibitorof tubulin polymerization that induces G2/M cell cycle arrest and celldeath at low picomolar intracellular concentrations (Sapra et al., 2011,Expert Opin Investig Drugs 20(8):1131-49 and Turner et al., 1998,Prostate 34(3):175-81).

H661 lung cancer cells were seeded onto a 4-well chamber slide systemwith a CC2 coated growth surface (Thermo Scientific) and treated for 48hours with hu24-vc0101, negative control ADC, unconjugated hu24mAb at0-4 g/mL or free 0101 auristatin at 0.1-10 nM. The cells were then fixedin 3% paraformaldehyde, washed with PBS, permeabilized with 0.5%Triton-X® (Sigma Chemical) in PBS, washed with PBS, and incubated withblocking buffer (5% normal goat serum and 0.2% Tween-20 in PBS). Cellswere incubated with the primary anti-α-tubulin antibody (Sigma NoT9026,clone DM1A) in blocking buffer for 1 hour at room temperature.Afterwards, the cells were washed with PBS and incubated for 30 minuteswith an Alexa Fluor® 488-conjugated secondary antibody (Invitrogen Corp)and 4′,6-diamidino-2-phenylindole (DAPI) to stain the DNA. The cellswere visualized on a Zeiss LSM 510 Meta confocal microscope.

Treatment of cells with free 0101 or hu24-vc0101 ADC disruptedmicrotubule structure and led to G2/M cell cycle arrest. In contrast,neither unconjugated hu24 mAb nor negative control mAb elicited theseresponses (FIG. 22). These results demonstrate that hu24-vc0101 ADC canelicit cytotoxicity in an antigen-dependent manner by disruptingmicrotubule structure and causing G2/M arrest. This mechanism isconsistent with previous studies on unconjugated auristatins.

Example 15 Hu24-Vc0101 ADC Effect on Endothelial Cells

FIG. 23 shows that hu24-vc0101 ADC inhibits angiogenesis in a standardin vitro HUVEC sprouting assay. Briefly, HUVEC cells (Lonza-#CC-2517)were used to coat Cytodex beads (Sigma #C0646-5G) at a ratio ofapproximately 1×10⁶ cells/2500 beads, and then placed overnight in an37° C./5% CO2 incubator with Endothelial Cell Growth Medium (Lonza #CC-3162). The following day the beads were washed with Endothelial CellGrowth Medium, and re-suspended in a solution of a 2.0 mg/ml fibrinogentype I (filter sterilized) in DPBS and 0.15 units/ml Aprotinin. Intoeach well of a 24 well plate, 0.3125 units of thrombin were added priorto addition of 500 ul of the bead solution. To facilitate clotting, theplates were placed in a 37° C. incubator for 15 minutes. Lastly skinfibroblast cells (Detroit 551—ATCC #CCL-110), suspended in EndothelialCell Growth Medium, were carefully laid on top of the formed fibrinogenclot. The cells were fed, with their respective drug treatment, everyother day and grown for 8 days. The degree of HUVEC sprouting andformation of branching vessels was observed.

Hu24-vc0101 ADC inhibited sprouting angiogenesis in this assay at 1g/mL, while the negative control ADC did not. This result demonstratesthat the anti-PTK7 ADC can inhibit angiogenesis in a target-specificmanner

Example 16 Reduction of Tumor-Initiating Cells (TIC)

To determine whether anti-PTK7 antibody-drug conjugate treatmentsreduced tumor-initiating cell (TIC) frequency in tumors, BR13 TNBCbreast tumors were treated with 4 mg/kg hu24-vc0101 ADC, 4 mg/kg controlIgG ADC, or 20 mg/kg docetaxel chemotherapy twice weekly for a total of3 doses (Days 0, 3 & 7). Live, residual human tumor cells (i.e. hESA⁺mCD45⁻ mH-2Kd⁻) were isolated from dissociated tumors at day 10 andre-implanted into naïve animals in a limit dilution analysis (LDA).Resulting tumor incidence was monitored for up to 40 weekspost-transplant. The day of tumor harvest (day 10) and serialtransplantation was chosen based on when tumors were starting to regressfollowing hu24-vc0101 exposure. Tumors were dissociated and stained withanti-human ESA, anti-mouse CD45, and anti-mouse H-2Kd antibodies. Threetumors per treatment group were pooled, and live human tumor cells weresorted by flow cytometry. Groups of 10 mice were injected with either293, 73, 37 or 15 tumor cells sorted from control IgG ADC treatedtumors; 159, 90, 40 or 10 tumor cells sorted from hu24-vc0101 treatedtumors; or 257, 33 or 15 tumor cells sorted from docetaxel-treatedtumors. Tumors in recipient mice were measured weekly and tumors thatexceeded 200 mm³ in recipient mice were scored as positive. UsingPoisson distribution statistics, via L-Calc software (StemcellTechnologies, Vancouver, BC), injected cell doses of recipients with andwithout tumors by 40 weeks post-transplant were used to calculate thefrequency of TIC after each treatment.

The TIC frequency in hu24-vc0101 treated tumors was 5.5-fold lower thanin control IgG ADC treated tumors (p=0.0013; Table 38). The TICfrequency in docetaxel-treated tumors was 2.1-fold lower than in controlIgG ADC treated tumors (p=0.09; Table 38). In summary, mice injectedwith hu24-vc0101 treated tumor cells consistently produced less tumorsthan mice injected with similar number of control IgG ADC treated tumorcells, which indicated that hu24-vc0101 treatment specifically reducedTICs.

TABLE 38 Tumor-initiating cell (TIC) frequency in BR13 tumor model. #Cells # Animals p-value implanted with # Animals TIC relative toPre-treatment Group per animal tumors in group frequency control ADCControl ADC A1 293 9 9 1 in 71 Not (4 mpk) A2 73 6 8 applicable A3 37 38 A4 15 0 8 hu24-vc0101 B1 159 3 7 1 in 393 0.0013 (4 mpk) B2 90 2 8 B340 0 10 B4 10 0 8 Docetaxel C1 257 8 9 1 in 149 0.09 (20 mpk) C2 33 1 8C3 15 0 6

1. An antibody-drug conjugate of the formula: Ab-(L-D), wherein: (a) Abis an antibody, or antigen-binding fragment thereof, that binds to humanprotein tyrosine kinase 7 (PTK7) comprising: at least one of a heavychain variable region comprising three CDRs set forth as SEQ ID NOs: 3,7, and 11 and a light chain variable region comprising three CDRs setforth as SEQ ID NOs: 17, 19, and 21; or at least one of a heavy chainvariable region comprising three CDRs set forth as SEQ ID NOs: 27, 31,and 35 and a light chain variable region comprising three CDRs set forthas SEQ ID NOs: 41, 43, and 45; and (b) L-D is a linker-drug moiety,wherein L is a linker, and D is a drug comprising a calicheamicin. 2.-8.(canceled)
 9. The antibody-drug conjugate of claim 1, wherein theantibody comprises a heavy chain variable region having an amino acidsequence that is at least 90% identical to SEQ ID NO: 1 and a lightchain variable region having an amino acid sequence that is at least 90%identical to SEQ ID NO: 15 or a heavy chain variable region having anamino acid sequence that is at least 90% identical to SEQ ID NO: 25 anda light chain variable region having an amino acid sequence that is atleast 90% identical to SEQ ID NO:
 39. 10. The antibody-drug conjugate ofclaim 9, wherein the antibody comprises a heavy chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO: 1 and a lightchain variable region comprising an amino acid sequence set forth as SEQID NO: 15 or a heavy chain variable region comprising an amino acidsequence set forth as SEQ ID NO: 25 and a light chain variable regioncomprising an amino acid sequence set forth as SEQ ID NO:
 39. 11. Theantibody-drug conjugate of claim 10, wherein the antibody comprises anIgG heavy chain constant region.
 12. The antibody-drug conjugate ofclaim 11, wherein the antibody comprises a heavy chain comprising anamino acid sequence set forth as SEQ ID NO: 13 or a heavy chaincomprising an amino acid sequence set forth as SEQ ID NO:
 37. 13. Theantibody-drug conjugate of claim 10, wherein the antibody comprises akappa light chain constant region.
 14. The antibody-drug conjugate ofclaim 13, wherein the antibody comprises a light chain comprising anamino acid sequence set forth as SEQ ID NO: 23 or a light chaincomprising an amino acid sequence set forth as SEQ ID NO:
 47. 15. Theantibody-drug conjugate of claim 10, wherein the antibody comprises aheavy chain comprising an amino acid sequence set forth as SEQ ID NO: 13and a light chain comprising an amino acid sequence set forth as SEQ IDNO: 23 or a heavy chain comprising an amino acid sequence set forth asSEQ ID NO: 37 and a light chain comprising an amino acid sequence setforth as SEQ ID NO:
 47. 16.-49. (canceled)
 50. The antibody-drugconjugate of claim 1, wherein the antibody-drug conjugate has adrug-to-antibody ratio (DAR) of 1 to
 8. 51. The antibody-drug conjugateof claim 1, wherein the calicheamicin is N-acetyl-γ-calicheamicin. 52.The antibody-drug conjugate of claim 1, wherein the linker is (4(4′acetylphenoxy)butanoic acid)(AcBut) and wherein the drug isN-acetyl-γ-calicheamicin dimethyl hydrazide (CM).
 53. An antibody-drugconjugate of the formula: Ab-(L-D), wherein: (a) Ab is an antibody thatbinds to human protein tyrosine kinase 7 (PTK7) comprising a heavy chaincomprising an amino acid sequence set forth as SEQ ID NO: 13 and a lightchain comprising an amino acid sequence set forth as SEQ ID NO: 23 or aheavy chain comprising an amino acid sequence set forth as SEQ ID NO: 37and a light chain comprising an amino acid sequence set forth as SEQ IDNO: 47; and (b) L-D is a linker-drug moiety, wherein L is (4 (4(4′acetylphenoxy)butanoic acid)(AcBut) and wherein the drug isN-acetyl-γ-calicheamicin dimethyl hydrazide (CM); and wherein theantibody-drug conjugate has a drug-to-antibody ratio (DAR) of 1 to 8.54. A pharmaceutical composition comprising the antibody-drug conjugateof claim 53 and a pharmaceutically acceptable carrier.
 55. A compositioncomprising a plurality of the antibody-drug conjugate of claim 53, andoptionally a pharmaceutical carrier, wherein the composition has anaverage DAR within a range of 1 to
 8. 56. A process for producing theantibody-drug conjugate of claim 53, comprising: (a) linking AcBut toCM; (b) conjugating the linker-drug moiety to the antibody; and (c)purifying the antibody-drug conjugate.
 57. A method of treating aPTK7-associated disorder comprising administering a therapeuticallyeffective amount of a composition comprising the antibody-drug conjugateof claim 53 to a subject in need thereof.
 58. The method of claim 57,wherein the PTK7-associated disorder is a hyperproliferative disorder.59. The method of claim 58, wherein the hyperproliferative disorder is aneoplastic disorder.
 60. The method of claim 59, wherein the neoplasticdisorder is a solid tumor.
 61. The method of claim 60, wherein the solidtumor is selected from the group consisting of breast cancer, ovariancancer, colorectal cancer, esophageal cancer, gastric cancer, melanoma,sarcoma, kidney cancer, pancreatic cancer, prostate cancer, livercancer, and lung cancer.
 62. The method of claim 61, wherein the breastcancer is triple-negative breast cancer (TNBC).
 63. The method of claim61, wherein the lung cancer is small cell lung cancer (SCLC).
 64. Themethod of claim 61, wherein the cancer is ovarian cancer.